CN113347994B

CN113347994B - Treatment and prevention of cancer using HER3 antigen binding molecules

Info

Publication number: CN113347994B
Application number: CN202080009114.6A
Authority: CN
Inventors: J·D·博伊德-柯克普; 关思宇; P·英格拉姆; K·帕兹基维奇; V·桑塞农; D·萨克; 吴志浩
Original assignee: Hummingbird Biotechnology Pte Ltd
Current assignee: Hummingbird Biotechnology Pte Ltd
Priority date: 2019-09-11
Filing date: 2020-09-10
Publication date: 2022-09-23
Anticipated expiration: 2040-09-10
Also published as: JP2022548057A; EP3818086B1; BR112022004474A2; WO2021048274A1; CN113347994A; CA3149853A1; BR112022004474A8; AU2020347473A1; ES2896921T3; TW202123926A; US20210155712A1; GB201913079D0; US20220153870A1; DK3818086T3; CN116059347A; PL3818086T3; KR20220086554A; EP3943509A1; EP3818086A1; MX2022002977A

Abstract

Disclosed are methods of treating or preventing cancer in a subject, wherein the cancer comprises cells having a mutation that results in increased expression of a HER3 ligand, wherein the method comprises administering to the subject a therapeutically or prophylactically effective amount of a molecule capable of binding to HER3 antigen.

Description

Treatment and prevention of cancer using HER3 antigen binding molecules

The present application claims priority from GB 1913079.8 filed 2019, 9, 11, the contents and elements of which are incorporated herein by reference for all purposes.

Technical Field

The present invention relates to the field of molecular biology, more specifically to antibody technology and methods of treatment and prevention.

Background

Increased HER3 expression is associated with poor prognosis in a variety of solid tumors, including breast, gastric, head and neck, pancreatic, ovarian and lung cancers. HER 3-mediated signal transduction has an adverse effect on tumor progression; HER3 upregulation was associated with resistance to anti-HER 2 and anti-EGFR treatment, and solid tumors refractory to anti-PD-1 treatment showed higher HER3 expression compared to responders to anti-PD-1 treatment.

HER3 binding antibodies are described, for example, in Zhang et al, Acta Biochimica et Biophysica Sinica (2016)48 (1); 39-48. anti-HER 3 antibody LJM-716 binds to epitopes on subdomains II and IV of the extracellular domain of HER3, locking HER3 in an inactive conformation (Garner et al, Cancer Res (2013)73: 6024-and 6035). MM-121 (also known as sirtuimab (seribanumab)) has been shown to inhibit HER 3-mediated signal transduction by blocking the binding of regulatory protein (heregulin) (HRG) to HER3 (Schoeberl et al, sci. signal. (2009)2(77): ra 31). Palituzumab (Patritumab) (also known as U-1287 and AMG-888) also blocks binding of regulatory proteins to HER3 (see, e.g., Shimizu et al Cancer Chemother Pharmacol (2017)79(3): 489-495. RG7116 (also known as lumestruzumab (lumretuzumab)) and RO-5479599) recognizes epitopes in subdomain I of the extracellular domain of HER3 (see, e.g., Mirschberger et al Cancer research (2013)73(16)5183-5194) KTN3379 binds HER 34 by interacting with amino acid residues in subdomain III (corresponding to positions of SEQ ID NO: 1: Gly476, Pro477, Arg481, Gly452, Arg, Ser450, Gly420, Ala451, Gly419, Arg421, Thr394, AcArg 423, Arg 423, Lys, Gly 356, Pro477, Leu 203, Leu-5926, Leu-Asp 26, Leu-202, Pro 310. Met 27, Met 14, Leu-14, Leu-76, Met-14, Met-14, Met, Lys, Met-9, Met, Lys, Met, and Met, 25, Met, 25, III, and Met, and the like Eur J Cancer 2012; 48: 126). REGN1400 also inhibits ligand binding to HER3 (see Zhang et al, Mol Cancer Ther (2014)13: 1345-1355). RG7597 (agotuzumab) is a Dual Acting Fab (DAF) capable of binding both HER3 and EGFR, and binds subdomain III of HER3 (see Schaefer et al, Cancer Cell (2011)20(4): 472-. MM-111 and MM-141 are bispecific antibodies with a HER3 binding arm that inhibits binding of HRG ligands to HER3 (see se McDonagh et al. Mol Cancer Ther (2012)11: 582-.

Disclosure of Invention

The present invention provides an antigen binding molecule capable of binding to HER3 according to any of the embodiments described herein for use in a method of treatment or prevention of a cancer in a subject, wherein the cancer comprises a cell characterized by HER3 ligand expression/overexpression.

Also provided is the use of an antigen binding molecule capable of binding HER3 according to any one of the embodiments described herein in the manufacture of a medicament for use in a method of treatment or prevention of a cancer in a subject, wherein the cancer comprises a cell characterized by HER3 ligand expression/overexpression.

Also provided is a method of treating or preventing a cancer in a subject, wherein the cancer comprises cells characterized by HER3 ligand expression/overexpression, wherein the method comprises administering to the subject a therapeutically or prophylactically effective amount of an antigen binding molecule capable of binding to HER3 according to any of the embodiments described herein.

In some embodiments according to aspects of the invention, the cancer comprises cells comprising a mutation that results in increased expression of HER3 ligand.

More specifically, the invention provides the use of an antigen binding molecule capable of binding HER3 in a method of treating or preventing cancer in a subject, wherein the cancer comprises cells comprising a mutation that results in increased expression of HER3 ligand, and wherein the antigen binding molecule comprises:

(i) A heavy chain variable region (VH) comprising the following CDRs:

has the sequence shown in SEQ ID NO: 43 of the amino acid sequence HC-CDR1

Has the sequence shown in SEQ ID NO:46 of the amino acid sequence shown in HC-CDR2

Has the sequence shown in SEQ ID NO: 51, HC-CDR 3; and

(ii) a light chain variable region (VL) comprising the following CDRs:

LC-CDR1 having amino acid sequence shown in SEQ ID NO. 91

LC-CDR2 having amino acid sequence shown in SEQ ID NO. 94

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 99.

Also provided is the use of an antigen binding molecule capable of binding to HER3 in the manufacture of a medicament for use in a method of treating or preventing cancer in a subject, wherein the cancer comprises a cell comprising a mutation that results in increased expression of HER3 ligand, and wherein the antigen binding molecule comprises:

(i) a heavy chain variable region (VH) comprising the following CDRs:

has the sequence of SEQ ID NO: 43 of the amino acid sequence shown in SEQ ID NO: HC-CDR1

HC-CDR2 having amino acid sequence shown in SEQ ID NO. 46

Has the sequence of SEQ ID NO: 51, HC-CDR 3; and

(ii) a light chain variable region (VL) comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 91

LC-CDR2 having the amino acid sequence shown in SEQ ID NO 94

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 99.

Also provided is a method of treating or preventing cancer in a subject, wherein the cancer comprises cells comprising a mutation that results in increased expression of a HER3 ligand, wherein the method comprises administering to the subject a therapeutically or prophylactically effective amount of an antigen binding molecule capable of binding to HER3, and wherein the antigen binding molecule comprises:

(i) a heavy chain variable region (VH) comprising the following CDRs:

has the sequence shown in SEQ ID NO: 43 of the amino acid sequence shown in SEQ ID NO: HC-CDR1

HC-CDR2 having amino acid sequence shown in SEQ ID NO. 46

Has the sequence of SEQ ID NO: 51, HC-CDR 3; and

(ii) a light chain variable region (VL) comprising the following CDRs:

LC-CDR1 having amino acid sequence shown in SEQ ID NO. 91

LC-CDR2 having amino acid sequence shown in SEQ ID NO. 94

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 99.

In some embodiments according to aspects of the invention, the ligand of HER3 comprises an amino acid sequence having at least 60% sequence identity to the EGF-like domain of NRG.

In some embodiments, the cancer comprises a cell with NRG gene fusion. In some embodiments, the NRG gene fusion is selected from the group consisting of CLU-NRG1, CD74-NRG1, DOC4-NRG1, SLC3A2-NRG1, RBPMS-NRG1, WRN-NRG1, SDC4-NRG1, RAB2IL1-NRG1, VAMP2-NRG1, KIF13B-NRG1, THAP7-NRG1, SMAD4-NRG1, MDK-NRG1, TNC-NRG1, DIP2B-NRG1, MRPL 1-NRG1, PARP 1-NRG1, ROCK1-NRG1, DPYSL 1-NRG1, ATP1B1-NRG1, NRH 1-NRG1, CDNRG 1-NRG1, CDAK-NRG 1, CDK-NRG 1, CDNRG 1, CDAK 1-NRG1, CDK-NRG 1, CDNRG 1-NRG1, CDNRG 1-NRG1, CDNRG 1, CDK-NRG 1, CDNRG 1, CDK-NRG 1, CDNRG 1, CDK-NRG 1, CDNRG 1, CDK-NRG 1, CDNRG 1, CDK-NRG 1, CDNRG 1, CDK-NRG 1, CDK-NRG, BMPRIB-NRG1, TNFRSF10B-NRG1, MCPH1-NRG1 and SLC12A2-NRG 2. In some embodiments, the NRG gene fusion is selected from CLU-NRG1, CD74-NRG1, SLC3A2-NRG1, or VAMP2-NRG 1.

In some embodiments, the cancer is derived from: lung, breast, head, neck, kidney, ovary, pancreas, prostate, uterus, gall bladder, colon, rectum, bladder, soft tissue or nasopharynx.

In some embodiments, the cancer is selected from lung cancer, non-small cell lung cancer, lung adenocarcinoma, aggressive lung mucinous adenocarcinoma, lung squamous cell carcinoma, breast cancer, malignant epithelial carcinoma of the breast, invasive carcinoma of the breast, head and neck cancer, squamous cell carcinoma of the head and neck, kidney cancer, clear cell carcinoma of the kidney, ovarian cancer, serous cystadenocarcinoma of the ovary, pancreatic cancer, pancreatic ductal adenocarcinoma of the pancreas, prostate cancer, malignant epithelial carcinoma of the prostate, endometrial cancer, uterine carcinosarcoma, gallbladder cancer, cholangiocarcinoma, colorectal cancer, bladder cancer, urothelial bladder cancer, sarcoma, soft tissue sarcoma, neuroendocrine tumor, and neuroendocrine tumor of the nasopharynx. In some embodiments, the cancer is selected from lung cancer, non-small cell lung cancer, lung adenocarcinoma, invasive lung mucinous adenocarcinoma, and lung squamous cell carcinoma.

In some embodiments, the antigen binding molecule comprises:

(i) a VH region comprising the following CDRs:

HC-CDR1 having amino acid sequence shown in SEQ ID NO. 41

HC-CDR2 having the amino acid sequence shown in SEQ ID NO. 45

HC-CDR3 having the amino acid sequence shown in SEQ ID NO. 48; and

(ii) a VL region comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 88

LC-CDR2 having the amino acid sequence shown in SEQ ID NO. 92

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 95.

In some embodiments, the antigen binding molecule comprises:

a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO 36; and

comprising a VL region whose amino acid sequence has at least 70% sequence identity to the amino acid sequence shown in SEQ ID NO 83.

In some embodiments, the antigen binding molecule comprises:

a VH region comprising the following Framework Regions (FR):

HC-FR1 having amino acid sequence shown by SEQ ID NO. 53

HC-FR2 having amino acid sequence shown in SEQ ID NO. 59

HC-FR3 having amino acid sequence shown in SEQ ID NO. 66

HC-FR4 having the amino acid sequence shown in SEQ ID NO: 71.

In some embodiments, the antigen binding molecule comprises:

a VL region comprising the following framework regions:

LC-FR1 having amino acid sequence shown in SEQ ID NO. 104

LC-FR2 having amino acid sequence shown in SEQ ID NO. 110

LC-FR3 having the amino acid sequence shown in SEQ ID NO. 120

LC-FR4 having the amino acid sequence shown in SEQ ID NO. 125.

In some embodiments, the antigen binding molecule comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO 171.

In some embodiments, the antigen binding molecule comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 177.

Description of the preferred embodiment

The known anti-HER 3 antibodies fall broadly into two classes. The first type of antibody binds to domain I and/or III of HER3, thereby competitively inhibiting ligand binding to HER 3. Serratizumab (MM-121) is a representative member of this class, others include paliritumab (patritumab) (U3-1287 or AMG-888), lumtuzumab (lumretuzumab) (RG-7116), AV-203, GSK2849330, and REGN 1400. The second class of antibodies locks HER3 into an inactive conformation by binding to the interface between domains II and IV or between domains II and III. LJM-716 is a representative example of this type, as is KTN 3379.

The present invention relates to novel HER 3-binding molecules with improved properties compared to known anti-HER 3 antibodies.

The inventors resorted to targeting to generate antigen binding molecules that bind to a specific region of interest in the extracellular region of HER 3. The HER 3-binding molecules of the invention have a combination of desirable biophysical and/or functional properties compared to antigen binding molecules disclosed in the prior art.

In an embodiment of the invention, the antigen binding molecule is capable of binding to subdomain II (SEQ ID NO:16) of the extracellular region of HER3 and inhibiting the binding of the bound HER3 molecule to an interacting partner.

In particular, the HER3 binding antigen binding molecules described herein are demonstrated to bind to an epitope of HER3, providing (i) effective inhibition of binding of HER3 to interacting partners (e.g. EGFR, HER2) and (ii) high affinity binding to HER3 in the presence and absence of NRG ligand. This unique combination of properties provides strong inhibition of downstream signaling and exceptional anti-cancer activity against a variety of cancers.

HER3

HER3 (also known as, e.g., ERBB3 LCCS2, MDA-BF-1) is a protein identified as UniProt P21860. Alternative splicing of mRNA encoded by the human ERBB3 gene produces five different isoforms: isoform 1 (Unit: P21860-1, v 1; SEQ ID NO: 1); isoform 2 (Unit: P21860-2; SEQ ID NO:2) which comprises a sequence different from SEQ ID NO:1 from position 141 and lacks the amino acid sequence corresponding to positions 183-1342 of SEQ ID NO: 1; isoform 3 (Unit: P21860-3; SEQ ID NO:3) which comprises a C331F substitution relative to SEQ ID NO:1 and lacks the amino acid sequence corresponding to position 332-1342 of SEQ ID NO: 1; isoform 4 (Unit: P21860-4; SEQ ID NO:4) which lacks the amino acid sequence corresponding to positions 1-59 of SEQ ID NO: 1; and isoform 5 (Unit: P21860-5; SEQ ID NO:5), which lacks the amino acid sequence corresponding to positions 1-643 of SEQ ID NO: 1.

The N-terminal 19 amino acids of SEQ ID NO 1 to 3 constitute the signal peptide, so that the mature forms of

isoforms

1, 2 and 3 of HER3 (i.e.after treatment to remove the signal peptide) have the amino acid sequences shown in

SEQ ID NO

6, 7 and 8, respectively.

The structure and function of HER3 are described, for example, in Cho and Leahy Science (2002)297(5585):1330-1333, Singer et al, Journal of Biological Chemistry (2001)276, 44266-44274, Roskoski et al, Pharmacol. Res. (2014)79:34-74, Bazley and Gullick Endocrine-Related Cancer (2005) S17-S27 and Mujoo et al, Oncotarget (1023) 5 (2014)5(21): 10222-6, which are all incorporated herein by reference. HER3 is a single pass transmembrane ErbB receptor tyrosine kinase with an N-terminal extracellular region (SEQ ID NO:9) that includes two leucine-rich subdomains (domains I and III, shown in SEQ ID NO:15 and 17, respectively) and two cysteine-rich subdomains (domains II and IV, shown in SEQ ID NO:16 and 18, respectively). Domain II contains a beta hairpin loop dimer (SEQ ID NO:19) that participates in intermolecular interactions with other HER receptor molecules. The extracellular domain is linked to the cytoplasmic domain (SEQ ID NO:11) via a transmembrane domain (SEQ ID NO: 10). The cytoplasmic domain included the membrane-proximal segment (SEQ ID NO:12), the protein kinase domain (SEQ ID NO:13) and the C-terminal segment (SEQ ID NO: 14).

Signaling through HER3 involves receptor homo-dimerization (i.e. with other HER3 receptors) or hetero-dimerization (with other HER receptors, e.g. HER2) and subsequent autophosphorylation by the protein kinase domain of cytoplasmic tyrosine. Phosphorylated tyrosine residues recruit aptamer/effector proteins (e.g. Grb2 and phospholipase C γ (PLC γ), comprising src homology domain 2(SH2) or phosphotyrosine binding (PTB) domains.

Signaling through HER3 may be activated in a ligand-dependent or ligand-independent manner. In the absence of ligand, the HER3 receptor molecule is typically expressed on the cell surface as a monomer with a conformation that prevents receptor dimerization, with the dimerization loop of subdomain II in intramolecular contact with the pocket on subdomain IV. Binding of HER3 ligands such as Neuregulin (NRG), e.g. NRG1 (also known as heregulin, HRG) or NRG2 to subdomains I and III of the extracellular region causes a conformational change which results in exposure of the dimeric ring of subdomain II, facilitating receptor dimerization and signaling. Some Cancer-related mutations in HER3 may disrupt the interaction of subdomains II and IV required to form an inactive "closed" conformation, leading to constitutive presentation of dimeric loops and activation of HER 3-mediated signals in the absence of ligand binding (see, e.g., Jaiswal et al, Cancer Cell (2013)23 (5: 603-.

In the present specification, "HER 3" refers to HER3 from any species and includes HER3 isoforms, fragments, variants (including mutants) or homologs from any species.

As used herein, a "fragment," "variant," or "homolog" of a protein can optionally be characterized as having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of a reference protein (e.g., a reference isoform). In some embodiments, fragments, variants, isomers, and homologs of a reference protein can be characterized by the ability to perform a function performed by such reference protein.

A "fragment" generally refers to a portion of a reference protein. A "variant" generally refers to a protein having an amino acid sequence that comprises one or more amino acid substitutions, insertions, deletions, or other modifications relative to the amino acid sequence of a reference protein, but retains a substantial degree of sequence identity (e.g., at least 60%) to the amino acid sequence of the reference protein. "isoforms" generally refer to variants of a reference protein expressed from the same species as the species of the reference protein (e.g., isoforms 1 to 5 of HER3 are all isoforms of each other). "homolog" generally refers to a reference protein species compared to different species produced by different species variants. For example, human HER3 isoform 1(P21860-1, v 1; SEQ ID NO:1) and rhesus HER3(Uniprot: F7HEH3-1, v 2; SEQ ID NO:20) are homologs of each other. Homologs include orthologs.

A "fragment" of a reference protein can be any length (by number of amino acids), although it can optionally be at least 20% of the length of the reference protein (i.e., the protein from which the fragment is derived), and the maximum length can be 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein.

HER3 fragments may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200 amino acids and a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200 or 1300 amino acids.

In some embodiments, HER3 is an isoform, fragment, variant, or homologue of HER3 from a mammal (e.g., a primate (rhesus monkey, cynomolgus monkey, non-human primate or human) and/or rodent (e.g., rat or mouse) may optionally be characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of an immature or mature HER3 isoform from a given species (e.g., human).

The isoform, fragment, variant or homologue may optionally be a functional isoform, fragment, variant or homologue, e.g. having the functional property/activity of a reference HER3, such as human HER3 isoform 1, as determined by analysis of a suitable assay for the functional property/activity. For example, an isoform, fragment, variant or homologue of HER3 may show association with one or more of the following: HER2, NRG1 (type I, type II, type III, type IV, type V or type VI) or NRG2(α or β).

In some embodiments, HER3 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with one of SEQ ID NOs 1 to 8.

In some embodiments, a HER3 fragment comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to one of SEQ ID nos. 9 to 19 (e.g., one of 9, 16 or 19).

Regions of particular interest on target molecules

The antigen binding molecules of the present invention are specifically designed to target the region of HER3 of particular interest. In a two-step approach, targeted HER3 regions were selected based on predicted antigenicity, function, and safety analyses. Specific antibodies to the HER3 targeting region were then prepared using a peptide corresponding to the HER3 target region as an immunogen to generate specific monoclonal antibodies, which were subsequently screened to identify antibodies capable of binding to HER3 in their native state. This method provides precise control of the epitope of the antibody.

The antigen binding molecules of the present invention may be defined with reference to the region of HER3 to which they bind. The antigen binding molecules of the invention can bind to a specific region of interest of HER 3. In some embodiments, the antigen binding molecule may bind a linear epitope of HER3, which consists of a contiguous sequence of amino acids (i.e., an amino acid primary sequence). In some embodiments, the antigen binding molecule may bind to a conformational epitope of HER3 that consists of amino acids that are not contiguous in the amino acid sequence.

In some embodiments, the antigen binding molecules of the invention bind to HER 3. In some embodiments, the antigen binding molecule binds to the extracellular region of HER3 (e.g., the region shown in SEQ ID NO: 9). In some embodiments, the antigen binding molecule binds to subdomain II of the extracellular region of HER3 (e.g., the region shown in SEQ ID NO: 16).

In some embodiments, the antigen binding molecule binds to the region of HER3 shown in SEQ ID No. 229. In some embodiments, the antigen binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID No. 229. In some embodiments, the antigen binding molecule binds to the region of HER3 shown in SEQ ID NOs 230 and 231. In some embodiments, the antigen binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID NOs 230 and 231. In some embodiments, the antigen binding molecule binds to the HER3 region shown in SEQ ID No. 230. In some embodiments, the antigen binding molecule contacts one or more amino acid residues of the region of HER3 as set forth in SEQ ID No. 230. In some embodiments, the antigen binding molecule binds to the HER3 region shown in SEQ ID No. 231. In some embodiments, the antigen binding molecule contacts one or more amino acid residues of the region of HER3 as set forth in SEQ ID No. 231.

In some embodiments, the antigen binding molecule binds to the HER3 region shown in SEQ ID NO. 23. In some embodiments, the antigen binding molecule contacts one or more amino acid residues of the HER3 region shown in SEQ ID No. 23. In some embodiments, the antigen binding molecule binds to the HER3 region shown in SEQ ID NO 21. In some embodiments, the antigen binding molecule contacts one or more amino acid residues of the HER3 region shown in SEQ ID No. 21. In some embodiments, the antigen binding molecule binds to the region of HER3 shown in SEQ ID NO 19. In some embodiments, the antigen binding molecule contacts one or more amino acid residues of the region of HER3 as set forth in SEQ ID No. 19. In some embodiments, the antigen binding molecule binds to the region of HER3 shown in SEQ ID NO. 22. In some embodiments, the antigen binding molecule contacts one or more amino acid residues of the region of HER3 as set forth in SEQ ID No. 22.

In some embodiments, the antigen binding molecule does not bind to the region of HER3 corresponding to positions 260 to 279 of SEQ ID No. 1. In some embodiments, the antigen binding molecule does not contact amino acid residues corresponding to the region of HER3 from positions 260 to 279 of SEQ ID No. 1. In some embodiments, the antigen binding molecule does not bind to the HER3 region shown in SEQ ID NO. 23. In some embodiments, the antigen binding molecule does not contact the amino acid residues of the region of HER3 as set forth in SEQ ID NO. 23.

The region of the peptide/polypeptide to which the antibody binds can be determined by the skilled artisan using a variety of methods well known in the art, including X-ray co-crystal analysis of antibody-antigen complexes, peptide scanning, mutagenesis mapping, mass spectrometry hydrogen-deuterium exchange analysis, phage display, competition ELISA and proteolysis-based "protection" methods. Such methods are described, for example: gershoni et al, BioDrugs, 2007, 21 (3): 145-156, which is incorporated herein by reference in its entirety.

In some embodiments, the antigen binding molecule is capable of binding to the same region of HER3, or an overlapping region of HER3, and an antibody that binds to said HER3 region comprises a VL sequence from one of antibody clones 10D1, 10D1_ c75, 10D1_ c76, 10D1_ c77, 10D1_ c78v1, 10D1_ c78v2, 10D1_11B, 10D1_ c85v1, 10D1_ c85v2, 10D1_ c85o1, 10D1_ c85o2, 10D1_ c 1, 10a 1, 4-35-B364-3635-B1, or VH sequence described herein. In some embodiments, the antigen binding molecule is capable of binding the same region of HER3 or an overlapping region of HER3, and an antibody that binds said HER3 region comprises VH and VL sequences from one of antibody clones 10D1_ c89, 10D1_ c90, or 10D1_ c 91. In some embodiments, the antigen binding molecule is capable of binding the same region of HER3 or an overlapping region of HER3, and the antibody that binds the HER3 region comprises VH and VL sequences from antibody clone 10D1_ c 89.

As used herein, "peptide" refers to two or more amino acid monomers linked by peptide bonds. Peptides are typically in the range of about 2 to 50 amino acids in length. A "polypeptide" is a polymer chain of two or more peptides. Polypeptides are typically greater than about 50 amino acids in length.

In some embodiments, the antigen binding molecules of the invention are capable of binding to a polypeptide comprising SEQ ID NO: 1. 3, 4, 6 or 8 or a polypeptide consisting thereof.

In some embodiments, the antigen binding molecule is capable of binding to a polypeptide comprising SEQ ID NO: 9 or a polypeptide consisting of the amino acid sequence shown in figure 9. In some embodiments, the antigen binding molecule is capable of binding to a polypeptide comprising SEQ ID NO: 16 or a polypeptide consisting of the amino acid sequence shown in seq id no.

In some embodiments, the antigen binding molecule is capable of binding to a polypeptide comprising SEQ ID NO: 229 or a peptide/polypeptide consisting thereof. In some embodiments, the antigen binding molecule is capable of binding to a peptide/polypeptide comprising or consisting of the amino acid sequences shown in SEQ ID NOs 230 and 231. In some embodiments, the antigen binding molecule is capable of binding to a polypeptide comprising SEQ ID NO:230 or a peptide/polypeptide consisting thereof. In some embodiments, the antigen binding molecule is capable of binding to a polypeptide comprising SEQ ID NO: 231 or a peptide/polypeptide consisting thereof. In some embodiments, the antigen binding molecule is capable of binding to a polypeptide comprising SEQ ID NO:23 or a peptide/polypeptide consisting thereof. In some embodiments, the antigen binding molecule is capable of binding to a polypeptide comprising SEQ ID NO: 21 or a peptide/polypeptide consisting thereof. In some embodiments, the antigen binding molecule is capable of binding to a polypeptide comprising SEQ ID NO: 19 or a peptide/polypeptide consisting thereof. In some embodiments, the antigen binding molecule is capable of binding to a polypeptide comprising SEQ ID NO: 22 or a peptide/polypeptide consisting thereof.

In some embodiments, the antigen binding molecule is not capable of binding to a polypeptide consisting of SEQ ID NO: 1 from position 260 to position 279. In some embodiments, the antigen binding molecule is not capable of binding to a peptide consisting of the amino acid sequence set forth in SEQ ID NO. 23.

The ability of a given peptide/polypeptide to specifically bind to a given molecule can be determined by assays according to Methods known to those skilled in the art, including by ELISA, immunoblotting (e.g., western immunoblotting), immunoprecipitation, surface plasmon resonance (SPR; see, e.g., Hearty et al, Methods Mol Biol (2012)907:411-442) or biolayer interferometry (see, e.g., Lad et al, (2015) J Biomol Screen 20(4): 498-507).

In embodiments where the antigen binding molecule is capable of binding a peptide/polypeptide comprising a reference amino acid sequence, the peptide/polypeptide may comprise one or more additional amino acids at one or both termini of the reference amino acid sequence. In some embodiments, the peptide/polypeptide comprises, e.g., 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 5-10, 5-20, 5-30, 5-40, 5-50, 10-20, 10-30, 10-40, 10-50, 20-30, 20-40, or 20-50 additional amino acids at one or both ends of the reference amino acid sequence.

In some embodiments, the additional amino acids provided at one or both ends (i.e., the N-terminus and C-terminus) of the reference sequence correspond to the positions of the ends of the reference sequence in the HER3 amino acid sequence. For example, when the antigen binding molecule is capable of binding to a polypeptide comprising SEQ ID NO: 23 and the sequence shown in SEQ ID NO: 23, the two additional amino acids may be threonine and lysine, corresponding to the amino acid sequence shown in SEQ ID NO: 278 and 279 of 1.

In some embodiments, the antigen binding molecule is capable of binding a peptide/polypeptide bound by an antibody comprising VH and VL sequences of one of the antibody clones described herein: antibody clones 10D1, 10D1_ c75, 10D1_ c76, 10D1_ c77, 10D1_ c78v1, 10D1_ c78v2, 10D1_11B, 10D1_ c85v1, 10D1_ c85v2, 10D1_ c85o1, 10D1_ c85o2, 10D1_ c87, 10D1_ c89, 10D1_ c90, 10D1_ c91, 10D1_ c92, 10D1_ c93, 10a6, 4-35-B2, or 4-35-B4. In some embodiments, the antigen binding molecule is capable of binding a peptide/polypeptide bound by an antibody comprising VH and VL sequences of one of the following antibody clones: antibody clones 10D1_ c89, 10D1_ c90 or 10D1_ c 91. In some embodiments, the antigen binding molecule is capable of binding a peptide/polypeptide bound by an antibody comprising the VH and VL sequences of antibody clone 10D1_ c 89.

Antigen binding molecules

The present invention provides antigen binding molecules capable of binding to HER 3.

"antigen binding molecule" refers to molecules capable of binding a target antigen, including monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fv, scFv, Fab, scFab, F (ab') ₂ 、Fab ₂ Diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g., VhH), etc.) so long as they exhibit binding to the relevant target molecule.

The antigen binding molecules of the present invention include a moiety capable of binding to a target antigen. In some embodiments, the moiety capable of binding to a target antigen comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody capable of specifically binding to a target antigen. In some embodiments, the moiety capable of binding to the target antigen comprises or consists of an aptamer capable of binding to the target antigen, such as a nucleic acid aptamer (e.g., reviewed in Zhou and Rossi Nat Rev Drug Discov.201716 (3): 181-. In some embodiments, the moiety capable of binding to a target antigen comprises or consists of an antigen-binding peptide/polypeptide, such as a peptide aptamer, thioredoxin, a single antibody, an anticalin (anticalin), a Kunitz domain, an avimer, a desmin (knottin), a phenanthroimer (fynomer), an aurimer (atrimer), DARPin, an affibody, a nanobody (i.e., a single domain antibody (sdAb)), an affinin (affilin), an armadillo repeat protein (ArmRP), an OBody, or a fibronectin-reviewed in, for example, Reverdatto et al, Curr Top Med chem.2015; 15(12): 1082-.

The antigen binding molecules of the present invention typically comprise an antigen binding domain comprising a VH and a VL of an antibody capable of specifically binding a target antigen. The antigen binding domain formed by VH and VL may also be referred to herein as the Fv region.

The antigen binding molecule may be or may comprise an antigen binding polypeptide or antigen binding polypeptide complex. An antigen binding molecule may comprise more than one polypeptide that together form an antigen binding domain. The polypeptides may be associated covalently or non-covalently. In some embodiments, the polypeptide forms part of a larger polypeptide comprising the polypeptide (e.g., in the case of an scFv comprising VH and VL, or in the case of a scFab comprising VH-CH1 and VL-CL).

An antigen-binding molecule can refer to a non-covalent or covalent complex of more than one polypeptide (e.g., 2, 3, 4, 6, or 8 polypeptides), such as an IgG-like antigen-binding molecule comprising two heavy chain polypeptides and two light chain polypeptides.

The antigen binding molecules of the present invention can be designed and prepared by monoclonal antibody (mAb) sequences capable of binding to HER 3. Antigen-binding regions of antibodies, such as single chain variable fragments (scFv), Fab and F (ab') ₂ And (3) fragment. An "antigen-binding region" is any fragment of an antibody that is capable of binding to the specific target of a given antibody.

Antibodies typically comprise six complementarity determining region CDRs; three of the heavy chain Variable (VH) regions: HC-CDR1, HC-CDR2 and HC-CDR3, and three of the light chain Variable (VL) regions: LC-CDR1, LC-CDR2 and LC-CDR 3. Together, these six CDRs define the antigenic determinant of the antibody, i.e., the portion of the antibody that binds to the target antigen.

The VH and VL regions contain Framework Regions (FRs) flanking each CDR that provide the scaffold for the CDR. From N-terminus to C-terminus, the VH region comprises the following structure: n-terminal- [ HC-FR1] - [ HC-CDR1] - [ HC-FR2] - [ HC-CDR2] - [ HC-FR3] - [ HC-CDR3] - [ HC-FR4] -C-terminal; the VL region comprises the structure: n-terminal- [ LC-FR1] - [ LC-CDR1] - [ LC-FR2] - [ LC-CDR2] - [ LC-FR3] - [ LC-CDR3] - [ LC-FR4] -C-terminal.

There are several different conventions for defining antibody CDRs and FRs, such as Kabat et al in "protein sequences of immunological interest" 5 th edition, national institutes of health, the United states public health service, Besserda, Maryland (1991), Chothia et al, journal of molecular biology 196: 901-917(1987), and VBASE2, such as Retter et al, Nucleic Acids Res (2005)33 (supplement 1): D671-D674. The CDRs and FRs of the VH and VL regions of the antibody clones described herein are defined according to the International IMGT (ImmunoGeneTiCs) information System (LeFranc et al, Nucleic Acids Res. (2015)43 (database album): D413-22) using the IMGT V-DOMAIN numbering convention as described by Lefranc et al, Dev. Comp. Immunol. (2003)27: 55-77.

In some embodiments, the antigen binding molecule comprises a CDR of the antigen binding molecule capable of binding HER 3. In some embodiments, the antigen binding molecule comprises an FR of an antigen binding molecule capable of binding HER 3. In some embodiments, the antigen binding molecule comprises CDRs and FRs of the antigen binding molecule capable of binding HER 3. That is, in some embodiments, the antigen binding molecule comprises a VH region and a VL region of the antigen binding molecule capable of binding HER 3.

In some embodiments, the antigen binding molecule comprises a VH region and a VL region derived or derived from an antibody clone that binds to HER3 described herein (i.e., an anti-HER 3 antibody clone 10D1_ c75, 10D1_ c76, 10D1_ c77, 10D1_ c78v1, 10D1_11B, 10D1_ c85v1, 10D1_ c85o1, 10D1_ c 1, 10D1 c 1, 4-35-1B, 4-1B-c 1, or a VL region such as VH region 1 c 1, e.g., VH region 1/1, 10D1 c 1, e.g., VH region 1, 1 c 1, e.g., VH region 1, 10D1 c 1, 1 c 1B, e.g., a.

In some embodiments, the antigen binding molecule comprises a VH region of any one of (1) to (10):

(1) (10D 1-derived) VH region comprising the following CDRs:

HC-CDR1 having the amino acid sequence shown in SEQ ID NO. 43

HC-CDR2 having the amino acid sequence shown in SEQ ID NO. 46

HC-CDR3 having the amino acid sequence shown in SEQ ID NO. 51,

or a variant thereof, wherein one or two or three of one or more of HC-CDR1, HC-CDR2 or HC-CDR3 is substituted with another amino acid.

(2) (10D1, 10D1_ c75, 10D1_ c76, 10D1_ c77, 10D1_ c78v1, 10D1_ c78v2, 10D1_11B, 10D1_ c87, 10D1_ c92, 10D1_ c93) VH regions comprising the following CDRs:

HC-CDR1 having the amino acid sequence shown in SEQ ID NO. 41

HC-CDR2 having the amino acid sequence shown in SEQ ID NO. 44

HC-CDR3 having the amino acid sequence shown in SEQ ID NO. 47,

(3) (10D1_ c85v1, 10D1_ c85v2) VH regions comprising the following CDRs:

HC-CDR1 having the amino acid sequence shown in SEQ ID NO. 41

HC-CDR2 having the amino acid sequence shown in SEQ ID NO. 45

HC-CDR3 having the amino acid sequence shown in SEQ ID NO. 47,

or a variant thereof, wherein one or two or three of the one or more HC-CDR1, HC-CDR2 or HC-CDR3 are substituted with another amino acid.

(4) (10D1_ c85o1) VH region comprising the following CDRs:

HC-CDR1 having amino acid sequence shown in SEQ ID NO. 41

HC-CDR2 having amino acid sequence shown in SEQ ID NO. 45

HC-CDR3 having the amino acid sequence shown in SEQ ID NO. 49,

(5) (10D1_ c85o2) VH region comprising the following CDRs:

HC-CDR1 having amino acid sequence shown in SEQ ID NO. 41

HC-CDR2 having amino acid sequence shown in SEQ ID NO. 45

HC-CDR3 having the amino acid sequence shown in SEQ ID NO. 50,

(6) (10D1_ c89, 10D1_ c90) VH regions comprising the following CDRs:

HC-CDR1 having amino acid sequence shown in SEQ ID NO. 41

HC-CDR2 having the amino acid sequence shown in SEQ ID NO. 45

HC-CDR3 having the amino acid sequence shown in SEQ ID NO. 48,

(7) (10D1_ c91) VH regions comprising the following CDRs:

HC-CDR1 having the amino acid sequence shown in SEQ ID NO. 42

HC-CDR2 having amino acid sequence shown in SEQ ID NO. 45

HC-CDR3 having the amino acid sequence shown in SEQ ID NO. 48,

(8) (10a6) VH regions comprising the following CDRs:

HC-CDR1 having the amino acid sequence shown in SEQ ID NO. 158

HC-CDR2 having the amino acid sequence shown in SEQ ID NO. 159

HC-CDR3 having the amino acid sequence shown in SEQ ID NO. 160,

(9) (4-35-B2) VH regions comprising the following CDRs:

HC-CDR1 having the amino acid sequence shown in SEQ ID NO. 128

HC-CDR2 having the amino acid sequence shown in SEQ ID NO. 129

HC-CDR3 having the amino acid sequence shown in SEQ ID NO. 130,

(10) (4-35-B4) VH regions comprising the following CDRs:

HC-CDR1 having amino acid sequence shown in SEQ ID NO. 144

HC-CDR2 having amino acid sequence shown in SEQ ID NO. 145

HC-CDR3 having the amino acid sequence shown in SEQ ID NO. 146,

In some embodiments, the antigen binding molecule comprises a VH region of any one of (11) to (24):

((11) (10D1) comprising the VH regions of the following FRs:

HC-FR1 having amino acid sequence shown in SEQ ID NO. 55

HC-FR2 having the amino acid sequence shown in SEQ ID NO. 58

HC-FR3 having amino acid sequence shown in SEQ ID NO. 69

HC-FR4 having the amino acid sequence shown by SEQ ID NO. 73,

or a variant thereof wherein one or two or three amino acids of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are substituted with another amino acid.

((12) (10D1_ c75, 10D1_ c92) VH regions comprising the following FRs:

HC-FR1 having amino acid sequence shown in SEQ ID NO. 52

HC-FR2 having amino acid sequence shown in SEQ ID NO. 56

HC-FR3 having amino acid sequence shown in SEQ ID NO. 61

HC-FR4 having the amino acid sequence shown by SEQ ID NO. 70,

or a variant thereof, wherein one or two or three amino acids of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are substituted with another amino acid.

((13) (10D1_ c76, 10D1_ c77, 10D1_ c78v1) VH regions comprising the following FRs:

HC-FR1 having amino acid sequence shown in SEQ ID NO. 52

HC-FR2 having amino acid sequence shown in SEQ ID NO. 56

HC-FR3 having amino acid sequence shown in SEQ ID NO. 62

HC-FR4 having the amino acid sequence shown by SEQ ID NO. 70,

((14) (10D1_ c78v2) VH region comprising the following FR:

HC-FR1 having amino acid sequence shown in SEQ ID NO. 52

HC-FR2 having amino acid sequence shown in SEQ ID NO. 57

HC-FR3 having amino acid sequence shown in SEQ ID NO. 62

HC-FR4 having the amino acid sequence shown by SEQ ID NO. 70,

((15) (10D1_11B) VH region comprising the following FRs:

HC-FR1 having the amino acid sequence shown in SEQ ID NO. 224

HC-FR2 having amino acid sequence shown in SEQ ID NO. 60

HC-FR3 having the amino acid sequence shown in SEQ ID NO. 63

HC-FR4 having the amino acid sequence shown by SEQ ID NO. 70,

((16) (10D1_ c85v1) VH region comprising the following FRs:

HC-FR1 having amino acid sequence shown in SEQ ID NO. 52

HC-FR2 having amino acid sequence shown in SEQ ID NO. 56

HC-FR3 having the amino acid sequence shown in SEQ ID NO. 64

HC-FR4 having the amino acid sequence shown by SEQ ID NO. 70,

((17) (10D1_ c85v2, 10D1_ c85o1, 10D1_ c85o2) VH regions comprising the following FRs:

HC-FR1 having amino acid sequence shown in SEQ ID NO. 52

HC-FR2 having amino acid sequence shown in SEQ ID NO. 57

HC-FR3 having the amino acid sequence shown in SEQ ID NO. 64

HC-FR4 having the amino acid sequence shown by SEQ ID NO. 70,

((18) (10D1_ c87, 10D1_ c93) VH regions comprising the following FRs:

HC-FR1 having amino acid sequence shown in SEQ ID NO. 52

HC-FR2 having amino acid sequence shown in SEQ ID NO. 56

HC-FR3 having amino acid sequence shown in SEQ ID NO. 65

HC-FR4 having the amino acid sequence shown by SEQ ID NO. 70,

((19) (10D1_ c89) VH region comprising the following FRs:

HC-FR1 having amino acid sequence shown by SEQ ID NO. 53

HC-FR2 having amino acid sequence shown in SEQ ID NO. 59

HC-FR3 having amino acid sequence shown in SEQ ID NO. 66

HC-FR4 having the amino acid sequence shown by SEQ ID NO:71,

((20) (10D1_ c90) VH region comprising the following FRs:

HC-FR1 having amino acid sequence shown in SEQ ID NO. 54

HC-FR2 having amino acid sequence shown in SEQ ID NO. 59

HC-FR3 having amino acid sequence shown by SEQ ID NO. 67

HC-FR4 having the amino acid sequence shown by SEQ ID NO:71,

((21) (10D1_ c91) VH region comprising the following FRs:

HC-FR1 having amino acid sequence shown by SEQ ID NO. 53

HC-FR2 having amino acid sequence shown in SEQ ID NO. 59

HC-FR3 having amino acid sequence shown in SEQ ID NO. 68

HC-FR4 having the amino acid sequence shown by SEQ ID NO. 72,

((22) (10A6) comprising the VH regions of the following FRs:

HC-FR1 having amino acid sequence shown by SEQ ID NO. 161

HC-FR2 having amino acid sequence shown in SEQ ID NO. 162

HC-FR3 having amino acid sequence shown by SEQ ID NO. 163

HC-FR4 having the amino acid sequence shown by SEQ ID NO. 73,

((23) (4-35-B2) VH regions comprising the following FRs:

HC-FR1 having amino acid sequence shown in SEQ ID NO. 131

HC-FR2 having amino acid sequence shown in SEQ ID NO. 132

HC-FR3 having amino acid sequence shown in SEQ ID NO. 133

HC-FR4 having the amino acid sequence shown by SEQ ID NO:134,

((24) (4-35-B4) comprising the VH regions of the following FRs:

HC-FR1 having amino acid sequence shown in SEQ ID NO:147

HC-FR2 having amino acid sequence shown in SEQ ID NO. 148

HC-FR3 having amino acid sequence shown in SEQ ID NO:149

HC-FR4 having the amino acid sequence shown by SEQ ID NO. 73,

In some embodiments, the antigen binding molecule comprises a VH region comprising a CDR of any one of (1) to (10) above and a FR of any one of (11) to (24) above.

In some embodiments, the antigen binding molecule comprises a VH region of any one of (25) to (41):

(25) a VH region comprising the CDR of (1) and the FR of (11), (12), (13), (14), (15), (16), (17), (18), (19), (20) or (21).

(26) A VH region comprising the CDR of (2) and the FR of (11).

(27) A VH region comprising the CDR of (2) and the FR of (12).

(28) A VH region comprising the CDR of (2) and the FR of (13).

(29) A VH region comprising the CDR of (2) and the FR of (14).

(30) A VH region comprising the CDR of (2) and the FR of (15).

(31) A VH region comprising the CDR of (2) and the FR of (18).

(32) A VH region comprising the CDR of (3) and the FR of (16).

(33) A VH region comprising the CDR of (3) and the FR of (17).

(34) A VH region comprising the CDR of (4) and the FR of (17).

(35) A VH region comprising the CDR of (5) and the FR of (17).

(36) A VH region comprising the CDR of (6) and the FR of (19).

(37) A VH region comprising the CDR of (6) and the FR of (20).

(38) A VH region comprising the CDR of (7) and the FR of (21).

(39) A VH region comprising the CDR of (8) and the FR of (22).

(40) A VH region comprising the CDR of (9) and the FR of (23).

(41) A VH region comprising the CDR of (10) and the FR of (24).

In some embodiments, the antigen binding molecule comprises a VH region of any one of (42) to (61):

(42) a VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID No. 24.

(43) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID No. 25.

(44) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO. 26, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

(45) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO. 27.

(46) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO 28, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

(47) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO. 29, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

(48) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO 30, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

(49) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO. 31, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

(50) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO. 32, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

(51) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID No. 33.

(52) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO. 34, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

(53) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO. 35, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

(54) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO. 36.

(55) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO 37, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

(56) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO 38, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

(57) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO. 39, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

(58) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO 40, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

(59) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO. 127, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

(60) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO. 143, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

(61) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:157, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, the antigen binding molecule comprises a VL region of any one of (62) to (71):

(62) (10D 1-derived) VL regions comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 91

LC-CDR2 having the amino acid sequence shown in SEQ ID NO 94

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 99;

or a variant thereof, wherein one or two or three amino acids of one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

(63) (10D1, 10D1_ c75, 10D1_ c78v1, 10D1_ c78v2, 10D1_11B, 10D1_ c87, 10D1_ c89, 10D1_ c91, 10D1_ c93) VL regions comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 88

LC-CDR2 having the amino acid sequence shown in SEQ ID NO. 92

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 95;

or a variant thereof, wherein one or two or three amino acids of one or more of LC-CDR1, LC-CDR2, or LC-CDR3 is substituted with another amino acid.

(64) (10D1_ c76) VL regions comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 89

LC-CDR2 having the amino acid sequence shown in SEQ ID NO. 92

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 95;

(65) (10D1_ c77) VL region comprising the following CDRs:

LC-CDR1 having amino acid sequence shown in SEQ ID NO. 90

LC-CDR2 having the amino acid sequence shown in SEQ ID NO. 92

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 96;

(66) (10D1_ c85v1, 10D1_ c85v2, 10D1_ c85o1, 10D1_ c85o2) VL regions comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 88

LC-CDR2 having the amino acid sequence shown in SEQ ID NO. 93

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 95;

(67) (10D1_ c90) VL regions comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 88

LC-CDR2 having the amino acid sequence shown in SEQ ID NO. 92

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 97;

(68) (10D1_ c92) VL regions comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 88

LC-CDR2 having the amino acid sequence shown in SEQ ID NO. 92

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 98;

(69) (10a6) a VL region comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 165

LC-CDR2 having the amino acid sequence shown in SEQ ID NO 166

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 167;

(70) (4-35-B2) a VL region comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 136

LC-CDR2 having amino acid sequence shown in SEQ ID NO. 137

LC-CDR3 having the amino acid sequence set forth in SEQ ID NO. 138;

(71) (4-35-B4) a VL region comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 151

LC-CDR2 having the amino acid sequence shown in SEQ ID NO. 152

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 153;

In some embodiments, the antigen binding molecule comprises a VL region of any one of (72) to (86):

(72) (10D1) a VL region comprising the following FRs:

LC-FR1 having amino acid sequence shown in SEQ ID NO 106

LC-FR2 having amino acid sequence shown in SEQ ID NO. 113

LC-FR3 having amino acid sequence shown in SEQ ID NO. 123

LC-FR4 having the amino acid sequence shown in SEQ ID NO. 126,

or a variant thereof, wherein one or two or three amino acids of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are substituted with another amino acid.

(73) (10D1_ c75) VL region comprising the following FRs:

LC-FR1 having amino acid sequence shown in SEQ ID NO. 100

LC-FR2 having amino acid sequence of SEQ ID NO. 107

LC-FR3 having amino acid sequence shown in SEQ ID NO. 114

LC-FR4 having the amino acid sequence shown in SEQ ID NO:124,

(74) (10D1_ c76) a VL region comprising the following FRs:

LC-FR1 having amino acid sequence shown in SEQ ID NO. 101

LC-FR2 having amino acid sequence shown in SEQ ID NO. 108

LC-FR3 having the amino acid sequence shown in SEQ ID NO. 115

LC-FR4 having the amino acid sequence shown in SEQ ID NO:124,

(75) (10D1_ c77) a VL region comprising the following FRs:

LC-FR1 having amino acid sequence shown in SEQ ID NO. 102

LC-FR2 having amino acid sequence shown in SEQ ID NO. 108

LC-FR3 having the amino acid sequence shown in SEQ ID NO. 116

LC-FR4 having the amino acid sequence shown in SEQ ID NO:124,

(76) (10D1_ c78v1, 10D1_ c78v2, 10D1_11B) VL region comprising the following FR:

LC-FR1 having amino acid sequence shown in SEQ ID NO. 103

LC-FR2 having amino acid sequence shown in SEQ ID NO. 108

LC-FR3 having amino acid sequence shown in SEQ ID NO. 117

LC-FR4 having the amino acid sequence shown in SEQ ID NO:124,

(77) (10D1_ c85v1, 10D1_ c85v2, 10D1_ c85o1, 10D1_ c85o2) VL regions comprising the following FRs:

LC-FR1 having amino acid sequence shown in SEQ ID NO. 103

LC-FR2 having amino acid sequence shown in SEQ ID NO. 108

LC-FR3 having the amino acid sequence shown in SEQ ID NO. 118

LC-FR4 having the amino acid sequence shown in SEQ ID NO:124,

(78) (10D1_ c87) VL region comprising the following FRs:

LC-FR1 having amino acid sequence shown in SEQ ID NO. 103

LC-FR2 having amino acid sequence shown in SEQ ID NO. 109

LC-FR3 having amino acid sequence shown in SEQ ID NO. 119

LC-FR4 having the amino acid sequence shown in SEQ ID NO:124,

(79) (10D1_ c89) VL region comprising the following FRs:

LC-FR1 having amino acid sequence shown in SEQ ID NO. 104

LC-FR2 having amino acid sequence shown in SEQ ID NO. 110

LC-FR3 having amino acid sequence shown in SEQ ID NO. 120

Has the sequence of SEQ ID NO: 125, or a pharmaceutically acceptable salt thereof, and LC-FR4,

(80) (10D1_ c90) VL region comprising the following FRs:

LC-FR1 having amino acid sequence shown in SEQ ID NO. 105

LC-FR2 having amino acid sequence shown in SEQ ID NO. 110

LC-FR3 having amino acid sequence shown in SEQ ID NO. 121

LC-FR4 having the amino acid sequence shown in SEQ ID NO:124,

(81) (10D1_ c91) a VL region comprising the following FRs:

LC-FR1 having amino acid sequence shown in SEQ ID NO. 104

LC-FR2 having amino acid sequence shown in SEQ ID NO. 111

LC-FR3 having amino acid sequence shown in SEQ ID NO. 122

LC-FR4 having an amino acid sequence shown in SEQ ID NO. 125,

(82) (10D1_ c92) a VL region comprising the following FRs:

LC-FR1 having amino acid sequence shown in SEQ ID NO. 100

LC-FR2 having amino acid sequence shown in SEQ ID NO. 112

LC-FR3 having amino acid sequence shown in SEQ ID NO. 114

LC-FR4 having the amino acid sequence shown in SEQ ID NO:124,

(83) (10D1_ c93) VL region comprising the following FRs:

LC-FR1 having amino acid sequence shown in SEQ ID NO. 103

LC-FR2 having amino acid sequence shown in SEQ ID NO. 108

LC-FR3 having amino acid sequence shown in SEQ ID NO. 119

LC-FR4 having the amino acid sequence shown in SEQ ID NO:124,

(84) (10A6) a VL region comprising the following FRs:

LC-FR1 having amino acid sequence shown in SEQ ID NO. 168

LC-FR2 having the amino acid sequence shown in SEQ ID NO 169

LC-FR3 having amino acid sequence shown in SEQ ID NO. 170

LC-FR4 having the amino acid sequence shown in SEQ ID NO:142,

(85) (4-35-B2) a VL region comprising the following FRs:

LC-FR1 having amino acid sequence shown in SEQ ID NO. 139

LC-FR2 having amino acid sequence shown in SEQ ID NO. 140

LC-FR3 having amino acid sequence shown in SEQ ID NO. 141

LC-FR4 having the amino acid sequence shown in SEQ ID NO:142,

(86) (4-35-B4) VL region comprising the following FRs:

LC-FR1 having amino acid sequence shown in SEQ ID NO. 154

LC-FR2 having amino acid sequence represented by SEQ ID NO. 155

LC-FR3 having amino acid sequence shown in SEQ ID NO. 156

LC-FR4 having the amino acid sequence shown in SEQ ID NO:142,

In some embodiments, the antigen binding molecule comprises a VL region comprising a CDR recited in any one of (62) to (71) and a FR recited in any one of (72) to (86) above.

In some embodiments, the antigen binding molecule comprises a VL region of any one of (87) to (102):

(87) a VL region comprising the CDR of (62) and the FR of (72), (73), (74), (75), (76), (77), (78), (79), (80), (81) or (83).

(88) A VL region comprising the CDR of (63) and the FR of (72).

(89) A VL region comprising the CDR of (63) and the FR of (73).

(90) A VL region comprising the CDR of (63) and the FR of (76).

(91) A VL region comprising the CDR of (63) and the FR of (78).

(92) A VL region comprising the CDR of (63) and the FR of (79).

(93) A VL region comprising the CDR of (63) and the FR of (81).

(94) A VL region comprising the CDR of (63) and the FR of (83).

(95) A VL region comprising the CDR of (64) and the FR of (74).

(96) A VL region comprising the CDR of (65) and the FR of (75).

(97) A VL region comprising the CDR of (66) and the FR of (77).

(98) A VL region comprising the CDR of (67) and the FR of (80).

(99) A VL region comprising the CDR of (68) and the FR of (82).

(100) A VL region comprising the CDR of (69) and the FR of (84).

(101) A VL region comprising the CDR of (70) and the FR of (85).

(102) A VL region comprising the CDR of (71) and the FR of (86).

In some embodiments, the antigen binding molecule comprises a VL region of any one of (103) to (119):

(103) a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO 74.

(104) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO 75.

(105) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO. 76.

(106) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO. 77.

(107) A VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 78, and more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

(108) A VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 79, and more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of one.

(109) A VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 80, and more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

(110) A VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 81 has at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

(111) A VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 82, and more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of one.

(112) A VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 83 have at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

(113) A VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 84, and more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

(114) A VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 85, and more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

(115) A VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 86, and more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of one sequence identity.

(116) A VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 87, and more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

(117) A VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 135, and more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of one.

(118) A VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 150, and more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of one.

(119) A VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 164, and more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, the antigen binding molecule comprises a VH region described in any one of (1) to (61) above and a VL region described in any one of (62) to (119) above.

In embodiments of the invention where one or more amino acids are substituted with another amino acid, the substitution may be a conservative substitution, for example according to the table below. In some embodiments, amino acids in the same block in the middle column are substituted. In some embodiments, amino acids in the same row in the rightmost column are substituted:

in some embodiments, the substitution can be functionally conservative. That is, in some embodiments, a substitution may not affect (or may not substantially affect) one or more functional properties (e.g., target binding) of the antigen binding molecule comprising the substitution, as compared to an equivalent unsubstituted molecule.

The VH and VL regions of the antigen-binding region of an antibody together constitute the Fv region. In some embodiments, the antigen binding molecules of the invention comprise or consist of an Fv region that binds HER 3. In some embodiments, the VH and VL regions of the Fv are provided as a single polypeptide connected by a linker region, i.e., a single chain Fv (scfv).

In some embodiments, the antigen binding molecules of the invention comprise one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from a heavy chain constant sequence of an IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM.

In some embodiments, the immunoglobulin heavy chain constant sequence is human immunoglobulin G1 constant (SEQ ID NO:171) (IGHG 1; UniProt: P01857-1, v 1). SEQ ID NO:171 form a CH1 region (SEQ ID NO: 172). SEQ ID NO:171 form a hinge region (SEQ ID NO: 173) between the CH1 and CH2 regions. SEQ ID NO:171 form a CH2 region (SEQ ID NO: 174). The amino acid sequence of SEQ ID NO:171 form a CH3 region (SEQ ID NO: 175).

Exemplary antigen binding molecules were prepared using pFUSE-CHIg-hG1, which contained the substitutions D356E, L358M (positions numbered according to EU numbering) in the CH3 region. The amino acid sequence of the CH3 region encoded by pFUSE-CHIg-hG1 is shown in SEQ ID NO: 176. It will be appreciated that further substitutions may be provided for the CH3 region in accordance with modifications to the Fc region of the antigen binding molecules described herein.

In some embodiments, the CH1 region comprises or consists of the sequence of SEQ ID No. 172, or a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID No. 172. In some embodiments, the CH1-CH2 hinge region comprises or consists of the sequence of SEQ ID No. 173, or a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID No. 173. In some embodiments, the CH2 region comprises or consists of the sequence of SEQ ID No. 174, or a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID No. 174. In some embodiments, the CH3 region comprises or consists of the sequence of SEQ ID No. 175 or 176, or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID No. 175 or 176.

In some embodiments, the antigen binding molecules of the invention comprise one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the immunoglobulin light chain constant sequence is human immunoglobulin kappa constant (SEQ ID NO:177) (IGKC; Ckappa; UniProt: P01834-1, v 2). In some embodiments, the immunoglobulin light chain constant sequence is a human immunoglobulin lambda constant (IGLC; Clambda), such as IGLC1, IGLC2, IGLC3, IGLC6, or IGLC 7. In some embodiments, the CL region comprises or consists of SEQ ID NO:177, or a sequence corresponding to SEQ ID NO:177 having an amino acid sequence identity of at least 60%, preferably at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

The VL and light chain Constant (CL) regions of the antigen binding region of an antibody, as well as the VH and heavy chain constant 1(CH1) regions, together comprise the Fab region. In some embodiments, the antigen binding molecule comprises a Fab region comprising VH, CH1, VL, and CL (e.g., ck or C λ). In some embodiments, the Fab region comprises a polypeptide comprising VH and CH1 (e.g., a VH-CH1 fusion polypeptide) and a polypeptide comprising VL and CL (e.g., a VL-CL fusion polypeptide). In some embodiments, the Fab region comprises a polypeptide comprising a VH and a CL (e.g., a VH-CL fusion polypeptide) and a polypeptide comprising a VL and a CH (e.g., a VL-CH1 fusion polypeptide); that is, in some embodiments, the Fab region is a CrossFab region. In some embodiments, the VH, CH1, VL, and CL regions of a Fab or CrossFab are provided as a single polypeptide connected by a linker region, i.e., as a single chain Fab (scfab) or a single chain CrossFab (sccrossfab).

In some embodiments, the antigen binding molecule of the invention comprises or consists of a Fab region that binds to HER 3.

In some embodiments, the antigen binding molecules described herein comprise or consist of an intact antibody that binds HER 3. As used herein, "whole antibody" refers to an antibody having a structure substantially similar to that of an immunoglobulin (Ig). Different classes of immunoglobulins and their structures are described, for example, in Schroeder and Cavacini J Allergy Clin Immunol. (2010)125(202) S41-S52, which are incorporated herein by reference in their entirety.

Immunoglobulin of type G (i.e., IgG) is a glycoprotein of about 150kDa, comprising two heavy chains and two light chains. From N-terminus to C-terminus, the heavy chain comprises a VH followed by a heavy chain constant region comprising three constant domains (CH1, CH2 and CH3), and similarly the light chain comprises a VL followed by a CL. Depending on the heavy chain, immunoglobulins can be classified as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. The light chain may be kappa (. kappa.) or lambda (. lamda.).

In some embodiments, the antigen binding molecules described herein comprise or consist of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM that bind HER 3.

In some embodiments, the antigen binding molecules of the invention bind to HER3 at least monovalent. Binding valency refers to the number of binding sites in an antigen binding molecule for a given antigenic determinant. Thus, in some embodiments, the antigen binding molecule comprises at least one site that binds HER 3.

In some embodiments, the antigen binding molecule comprises more than one binding site for HER3, e.g. 2, 3 or 4 binding sites. The binding sites may be the same or different. In some embodiments, the antigen binding molecule is, e.g., divalent, trivalent, or tetravalent relative to HER 3.

Some aspects of the invention relate to multispecific antigen-binding molecules. By "multispecific" is meant that the antigen binding molecule exhibits specific binding to more than one target. In some embodiments, the antigen binding molecule is a bispecific antigen binding molecule. In some embodiments, the antigen binding molecule comprises at least two different antigen binding domains (i.e., at least two antigen binding domains, e.g., comprising different VH and VL).

In some embodiments, the antigen binding molecule binds to HER3 and another target (e.g., an antigen different from HER 3), and is therefore at least bispecific. The term "bispecific" refers to an antigen binding molecule capable of specifically binding at least two different antigenic determinants.

It is understood that the antigen binding molecules (e.g., multispecific antigen binding molecules) of the present invention comprise antigen binding molecules capable of binding to a specific target of the antigen binding molecule. For example, antigen binding molecules capable of binding to antigens other than HER3 and HER3 may include: (i) an antigen binding molecule capable of binding to HER3, and (ii) an antigen binding molecule capable of binding to an antigen other than HER 3.

It is also understood that the antigen binding molecules (e.g., multispecific antigen binding molecules) of the present invention may comprise an antigen binding polypeptide or antigen binding polypeptide complex capable of binding to a specific target of the antigen binding molecule. For example, an antigen binding molecule of the invention may comprise, for example, (i) an antigen binding polypeptide complex capable of binding to HER3 comprising a light chain polypeptide (comprising the structure VL-CL) and a heavy chain polypeptide (comprising the structure VH-CH1-CH2-CH 3), and (ii) an antigen binding polypeptide complex capable of binding to an antigen other than HER3 comprising a light chain polypeptide (comprising the structure VL-CL) and a heavy chain polypeptide (comprising the structure VH-CH1-CH2-CH 3).

In some embodiments, the component antigen binding molecules of a larger antigen binding molecule (e.g., a multispecific antigen binding molecule) may be referred to as, for example, an "antigen binding domain" or "antigen binding region" of the larger antigen binding molecule.

In some embodiments, the antigen binding molecule comprises an antigen binding molecule capable of binding to HER3 and an antigen binding molecule capable of binding to an antigen other than HER 3. In some embodiments, the antigen other than HER3 is an immune cell surface molecule. In some embodiments, the antigen other than HER3 is a cancer cell antigen. In some embodiments, the antigen other than HER3 is a receptor molecule, such as a cell surface receptor. In some embodiments, the antigen other than HER3 is a cell signaling molecule, such as a cytokine, chemokine, interferon, interleukin, or lymphokine. In some embodiments, the antigen other than HER3 is a growth factor or hormone.

Cancer cell antigens are antigens expressed or overexpressed by cancer cells. The cancer cell antigen may be any peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. Expression of cancer cell antigens may be associated with cancer. The cancer cell antigen can be aberrantly expressed by the cancer cell (e.g., an abnormal localization of the antigen expressed by the cancer cell), or an abnormal structure can be expressed by the cancer cell. Cancer cell antigens may be capable of eliciting an immune response. In some embodiments, the antigen is expressed on the cell surface of the cancer cell (i.e., the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the antigen moiety bound to the antigen binding molecule described herein is displayed on the outer surface of a cancer cell (i.e., is extracellular). The cancer cell antigen may be a cancer-associated antigen. In some embodiments, the cancer cell antigen is an antigen whose expression correlates with the development, progression, or severity of symptoms of a cancer. The cancer-associated antigen may be associated with the cause or pathology of cancer, or may be aberrantly expressed as a result of cancer. In some embodiments, a cancer cell antigen is an antigen whose expression is upregulated (e.g., at the RNA and/or protein level) by a cancer cell, e.g., as compared to the expression level of a comparable non-cancer cell (e.g., a non-cancer cell derived from the same tissue/cell type). In some embodiments, the cancer-associated antigen may be preferentially expressed by cancer cells, but not by comparable non-cancer cells (e.g., non-cancer cells derived from the same tissue/cell type). In some embodiments, the cancer-associated antigen can be a mutated oncogene or a mutated cancer suppressor gene product. In some embodiments, the cancer-associated antigen may be the product of an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, a carcinoembryonic antigen, or a cell-surface glycolipid or glycoprotein.

In some embodiments, the antigen other than HER3 is an antigen expressed by a HER 3-associated cancer cell. A HER 3-related cancer may be a HER 3-expressing cancer (e.g., HER3 protein expressed on the cell surface); such cancers may be referred to as "HER 3 positive" cancers. HER 3-related cancers include cancers in which the expression of HER3 gene/protein is a severe risk factor for the development, progression or severity of symptoms and/or metastasis of the cancer, and/or is positively correlated therewith. HER 3-related cancers include Zhang et al, biochemical and biophysical report (2016)48 (1): 39-48 and Sithanandam and Anderson Cancer, Gene Ther (2008)15 (7): 413-448, which is herein incorporated by reference in its entirety. In some embodiments, the cancer associated with HER3 may be lung cancer (e.g. NSCLC), melanoma, breast cancer, pancreatic cancer, prostate cancer, ovarian cancer, gastric cancer, colon cancer, or oral cancer.

The immune cell surface molecule may be any peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid or fragment thereof expressed at or on the cell surface of an immune cell. In some embodiments, the portion of the immune cell surface molecule that binds to the antigen binding molecule of the invention is on the outer surface of the immune cell (i.e., extracellular). The immune cell surface molecule may be expressed on the cell surface of any immune cell. In some embodiments, the immune cell may be a cell of hematopoietic origin, such as a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocytes may be, for example, T cells, B cells, Natural Killer (NK) cells, NKT cells, or Innate Lymphoid Cells (ILC) or precursors thereof (e.g., thymocytes or precursor B cells). In some embodiments, the immune cell surface molecule can be a co-stimulatory molecule (e.g., CD28, OX40, 4-1BB, ICOS, or CD27) or a ligand thereof. In some embodiments, the immune cell surface molecule can be a checkpoint molecule (e.g., PD-1, CTLA-4, LAG-3, TIM-3, VISTA, TIGIT, or BTLA) or a ligand thereof.

The multispecific antigen-binding molecules of the present invention may be provided in any suitable form, for example, as described in Brinkmann and Kontermann MAbs (2017)9 (2): 182, 212, which are incorporated herein by reference in their entirety. Suitable forms include those described in Brinkmann and Kontermann MAbs (2017)9 (2): 182-212 of FIG. 2, e.g., IgG ₂ 、F(ab’) ₂ Or the CovX body; IgG or IgG-like molecules, e.g., IgG, chimeric IgG, κ λ body universal HC; CH1/CL fusion proteins, such as scFv2-CH1/CL, VHH2-CH 1/CL; "variable domain only" bispecific antigen binding molecules, such as tandem scfv (tafv), triabodies, diabodies (Db), dsDb, Db (kih), DART, scDB, dsFv-dsFv, tandAb, triabodies (triple heads), tandem dAb/VHH, tetravalent dAb VHH; non-Ig fusion proteins, e.g. scFv ₂ Albumin, scDb-albumin, taFv-toxin, minibody, DNL-Fab ₂ 、DNL-Fab ₂ -scFv、DNL-Fab ₂ -IgG-cytokines ₂ ImmTAC (TCR-scFv); modified Fc and CH3 fusion proteins, such as scFv-Fc (kih), scFv-Fc (CH3 charge pairing), scFv-Fc (EW-RVT), scFv-Fc (HA-TF), scFv-Fc (SEED body), taFv-Fc (kih), scFv-Fc (kih) -Fv, Fab-Fc (kih) -scFv, Fab-scFv-Fc (kih), Fab-scFv-Fc (BEAT), Fab-scFv-Fc (SEED body), DART-Fc, scFv-CH3(kih), TriFab; fc fusions, e.g. diabodies, scDb-Fc, taFv-Fc, scFv-Fc-scFv, HCAb-VHH, Fab-scFv-Fc, scFv ₄ -Ig、scFv ₂ -FcAb; CH3 fusions, such as diabodies, scDb-CH 3; IgE/IgM CH2 fusions such as scFv-EHD2-scFv, scFvMHD 2-scFv; fab fusion proteins, e.g. Fab-scFv (diabody), Fab-scFv ₂ (triabodies), Fab-Fv, Fab-dsFv, Fab-VHH, orthogonal Fab-Fab'; non-Ig fusion proteins, e.g. DNL-Fab ₃ 、DNL-Fab ₂ -scFv、DNL-Fab ₂ -IgG-cytokines ₂ (ii) a Asymmetric IgG or IgG-like molecules, such as IgG (kih), IgG (kih) universal LC, ZW1 IgG universal LC, Biclonics universal LC, CrossMab (kih), scFab-IgG (kih), Fab-scFab-IgG (kih), orthogonal Fab IgG (kih), DuetMab, CH3 charge pairing + CH1/CL charge pairing, hinge/CH 3 charge pairing, SEED bodies, bispecific antibodies (duoblody), four-in-one CrossMab (kih), z-Y universal LC; LUZ-Y scFab-IgG, FcFc; additional and Fc-modified IgGs such as IgG (kih) -Fv, IgG HA-TF-Fv, IgG (kih) scFab, scFab-Fc (kih) -scFv2, scFab-Fc (kih) -scFv, semi DVD-Ig, DVI-Ig (four in one), CrossMab-Fab; modified Fc and CH3 fusion proteins, such as Fab-Fc (kih) -scFv, Fab-scFv-Fc (kih), Fab-scFv-Fc (BEAT), Fab-scFv-Fc-SEED bodies, TriFab; additional IgG-HC fusions, e.g., IgG-HC, scFv, IgG-dAb, IgG-taFV, IgG-CrossFab, IgG-orthogonal Fab, IgG- (C.alpha.C.beta.) Fab, scFv-HC-IgG, tandem Fab-IgG (orthogonal Fab) Fab-IgG (C.alpha.C.beta.Fab), Fab-IgG (CR3), Fab-hinge-IgG (CR 3); additional IgG-LC fusions, such as IgG-scFv (LC), scFv (LC) -IgG, dAb-IgG; additional IgG-HC and LC fusions, e.g. DVD-Ig, TVD-Ig, CODV-Ig, scFv ₄ IgG, Zy body (Zybody); fc fusionsFor example, Fab-scFv-Fc, scFv 4-Ig; f (ab') ₂ Fusions, e.g. F (ab') ₂ -scFv ₂ (ii) a CH1/CL fusion proteins, e.g. scFv ₂ -CH 1-hinge/CL; modified IgG, e.g. DAF (two-in-one IgG), Dutamab, Mab ² (ii) a And non-Ig fusions, e.g. DNL-Fab ₄ -IgG。

The skilled person is able to design and prepare bispecific antigen binding molecules. Methods for generating bispecific antigen-binding molecules include chemically cross-linking antigen-binding molecules or antibody fragments with, for example, reducible disulfide bonds or non-reducible thioether bonds, e.g., Segal and Bast, 2001, "generation of bispecific antigen-binding molecules", current methods of immunology, 14: IV: 2.13: 2.13.1-2.13.16, herein incorporated by reference in their entirety. For example, N-succinimidyl-3- (-2-pyridyldithio) -propionate (SPDP) can be used to chemically crosslink, e.g., Fab fragments via hinge region SH-groups, thereby producing disulfide-linked bispecific F (ab) ₂ A heterodimer.

Other methods of producing bispecific antigen binding molecules include the fusion of antibody-producing hybridomas with, for example, polyethylene glycol to make quadroma cells (quadroma cells) capable of secreting bispecific antibodies, e.g., d.m. and Bast, b.j.2001, "production of bispecific antigen binding molecules", current protocols in immunology, 14: IV: 2.13: 2.13.1-2.13.16.

Bispecific antigen-binding molecules of the invention may also be produced recombinantly by expression, e.g., of nucleic acid constructs encoding polypeptides of the antigen-binding molecules, e.g., antibody engineering: methods and protocols, second edition (Hough Press-Humana Press,2012), Chapter 40 "production of bispecific antigen binding molecules: diabodies and tandem scFv "(Hornig and

schwarz), or French, "how to prepare bispecific antigen binding molecules", Methods mol.med.2000; 40:333, 339, the entire contents of which are incorporated herein by reference. For example, two antigen binding fragments encoding the same can be prepared by molecular cloning techniquesThe light and heavy chain variable domains of a stretch (i.e., the light and heavy chain variable domains of an antigen-binding fragment capable of binding HER3, and the light and heavy chain variable domains of an antigen-binding fragment capable of binding another target protein) and includes DNA constructs that encode sequences for suitable linkers or dimerization domains between the antigen-binding fragments. The recombinant bispecific antibody can then be produced by expressing (e.g., in vitro) the construct in a suitable host cell (e.g., a mammalian host cell), and the expressed recombinant bispecific antibody can then optionally be purified.

Fc region

In some embodiments, the antigen binding molecules of the present invention comprise an Fc region.

In IgG, IgA, and IgD isotypes, the Fc region consists of the CH2 and CH3 regions of one polypeptide and the CH2 and CH3 regions of another polypeptide. The CH2 and CH3 regions from both polypeptides together form an Fc region. In IgM and IgE isotypes, the Fc region comprises three constant domains (CH2, CH3, and CH4), and CH2 through CH4 from two polypeptides together form the Fc region.

In preferred embodiments of the various aspects of the present disclosure, the Fc region comprises two polypeptides, each polypeptide comprising a CH2 region and a CH3 region.

In some embodiments, the antigen binding molecules of the present invention comprise an Fc region comprising modifications in one or more of the CH2 and CH3 regions that facilitate binding of the Fc region. Recombinant co-expression of the constituent polypeptides of the antigen-binding molecule and subsequent binding results in several possible combinations. To increase the yield of the desired combination of polypeptides in the antigen-binding molecule in recombinant production, it is advantageous to introduce a modification in the Fc region that promotes the binding of the desired combination of heavy chain polypeptides. The modifications can facilitate, for example, hydrophobic and/or electrostatic interactions between CH2 and/or CH3 regions of different polypeptide chains. Suitable methods are for example: ha et al, front. immunol (2016)7:394, herein incorporated by reference in its entirety.

In some embodiments, the antigen-antigen binding molecules of the invention comprise an Fc region comprising a paired substitution in the CH3 region of the Fc region in the form of one of the following, as shown in table 1 of Ha et al, front. KiH, KiHs-s, HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, SEED or A107.

In some embodiments, the Fc region comprises a "knob-in-hole" or "KiH" modification, for example as described in US7,695,936 and Carter, J Immunol Meth 248,7-15 (2001). In such embodiments, one CH3 region of the Fc region comprises a "knob" modification, while the other CH3 region comprises a "well" modification. The "knob" and "hole" modifications are located within the respective CH3 regions such that the "knob" can be located in the "hole" to promote heterodimerization (and inhibit homodimerization) of the polypeptide and/or to stabilize the heterodimer. Knobs are constructed by replacing amino acids with small (side) chains with amino acids with larger side chains (e.g. tyrosine or tryptophan). The pore is created by replacing an amino acid with a large side chain with an amino acid with a smaller side chain (e.g., alanine or threonine).

In some embodiments, one CH3 region of the Fc region of the antigen binding molecules of the invention comprises a T366W substitution (herein, the numbering of the positions/substitutions in the Fc, CH2 and CH3 regions is according to the EU numbering system described by Kabat et al, "Sequences of Proteins of Immunological Interest" (seq id No. 5, public health service, national institutes of health, bessel, maryland, 1991), and the other CH3 region of the Fc region comprises a Y407V substitution. In some embodiments, one of the CH3 regions of the Fc region of the antigen binding molecule comprises a T366W substitution, while the other CH3 region of the Fc region comprises a T366S and L368A substitution. In some embodiments, one CH3 region of the Fc region of the antigen binding molecule comprises a T366W substitution and the other CH3 region of the Fc region comprises Y407V, T366S and L368A substitutions.

In some embodiments, the Fc region comprises a "DD-KK" modification, for example as described in WO2014/131694a 1. In some embodiments, one CH3 region comprises K392D and K409D substitutions, while the other CH3 region of the Fc region comprises E356K and D399K substitutions. The modification promotes electrostatic interactions between the CH3 regions.

In some embodiments, the antigen binding molecules of the invention comprise a modified Fc region, referred to as a "bispecific antibody (Duobody)" format, as described in Labrijn et al, proceedings of the american academy of science (2013)110(13): 5145-50. In some embodiments, one CH3 region comprises the K409R substitution, while the other CH3 region of the Fc region comprises the K405L substitution.

In some embodiments, the antigen binding molecules of the invention comprise an Fc region comprising an "EEE-RRR" modification as described in Strop et al, J Mol Biol. (2012)420(3): 204-19. In some embodiments, one CH3 region comprises D221E, P228E, and L368E substitutions, and the other CH3 region of the Fc region comprises D221R, P228R, and K409R substitutions.

In some embodiments, the antigen binding molecule comprises an Fc region comprising "EW-RVT" modifications as described in Choi et al, Mol Cancer Ther (2013)12(12): 2748-59. In some embodiments, one CH3 region comprises K360E and K409W substitutions and the other CH3 region of the Fc region comprises Q347R, D399V and F405T substitutions.

In some embodiments, one CH3 region comprises the S354C substitution and the other CH3 region of the Fc region comprises the Y349C substitution. The introduction of these cysteine residues results in the formation of a disulfide bridge between the two CH3 regions of the Fc region, thereby further stabilizing the heterodimer (Carter (2001), J immunological Methods 248, 7-15).

In some embodiments, the Fc region comprises "KiH _S-S "modification". In some embodiments, one CH3 region comprises T366W and S354C substitutions, and the other CH3 region of the Fc region comprises T366S, L368A, Y407V, and Y349C substitutions.

In some embodiments, the antigen binding molecules of the invention comprise an Fc region comprising a "SEED" modification as described in Davis et al, Protein Eng Des Sel (2010)23(4): 195-Asa 202, wherein the β -segments of human IgG1 CH3 and IgA CH3 are interchanged.

In some embodiments, one CH3 region comprises S364H and F405A substitutions and the other CH3 region of the Fc region comprises Y349T and T394F substitutions (see, e.g., Moore et al, MAbs (2011)3(6): 546-57).

In some embodiments, one CH3 region comprises T350V, L351Y, F405A, and Y407V substitutions, and the other CH3 region of the Fc region comprises T350V, T366L, K392L, and T394W substitutions (see, e.g., Von Kreudenstein et al, MAbs (2013)5(5): 646-54).

In some embodiments, one CH3 region comprises K360D, D399M, and Y407A substitutions, and the other CH3 region of the Fc region comprises E345R, Q347R, T366V, and K409V substitutions (see, e.g., Leaver-Fay et al, Structure (2016)24(4): 641-51).

In some embodiments, One CH3 region comprises K370E and K409W substitutions and the other CH3 region of the Fc region comprises E357N, D399V, and F405T substitutions (see, e.g., Choi et al, PLoS One (2015)10(12): E0145349).

Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of Membrane Attack Complex (MAC), cell degranulation, production of cytokines and/or chemokines, and processing and presentation of antigens.

Modifications of the Fc region of antibodies that affect Fc-mediated functions are known in the art, for example in Wang et al, protein cells (2018)9(1):63-73, which are herein incorporated by reference in their entirety. Exemplary Fc region modifications known to affect antibody effector function are summarized in table 1 of Wang et al, protein cells (2018)9(1): 63-73.

Substituted combination F243L/R292P/Y300L/V305I/P396L in Stavenhagen et al, cancer research, (2007) describes that the substituted combination F243L/R292P/Y300L/V305I/P396L can increase binding to Fc γ RIIIa, thereby enhancing ADCC effect. Substituted combinations S239D/I332E or S239D/I332E/A330L are described in Lazar et al, Proc. Natl.Acad.Sci. (2006)103: 4005-. It is also described that a combination of S239D/I332E/a330L substitutions can reduce binding to Fc γ RIIb, thereby increasing ADCC effect. In Shields et al, J Biol Chem. (2001) 276: 6591-6604 describes that the substituted combination S298A/E333A/K334A increases binding to Fc γ RIIIa and thereby increases ADCC. The substitution combinations L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in one heavy chain and D270E/K326D/A330M/K334E in the other heavy chain increase binding to Fc γ RIIIa and thus ADCC, are described in Mimoto et al, MAbs (2013):5: 229-. The substituted combination G236A/S239D/I332E described in Richards et al, Mol Cancer Ther (2008)7: 2517-2527, increases binding to Fc γ RIIa and increases binding to Fc γ RIIIa, thereby increasing ADCP action.

Substituted combination K326W/E333S in Idusogene et al, J Immunol. (2001)166(4):2571-5 it is described that the substituted combination K326W/E333S increases the binding to C1q and thus the CDC effect. It is described in Moore et al, MAbs, (2010)2(2):181-9 that the substituted combination S267E/H268F/S324T can increase binding to C1q, thereby increasing CDC action. In Natsume et al, Cancer Res, (2008)68 (10): 3863-72, can increase binding to C1q and thereby increase CDC. In diebold et al, Science (2014)343 (6176): 1260-3, the substituted combination E345R/E430G/S440Y may increase hexamerization and thus CDC.

Substituted combination M252Y/S254T/T256E in Dall' Acqua et al, J Immunol. (2002) 169: 5171 the combination of substitutions M252Y/S254T/T256E described in 5180 increases the binding to FcRn at pH 6.0 and thus prolongs the half-life of the antigen binding molecule. In Zalevsky et al, Nat Biotechnol, (2010) 28: the combination of substitutions M428L/N434 described in 157-159 may increase binding to FcRn at pH 6.0, thereby extending the half-life of the antigen binding molecule.

When the heavy chain constant region/Fc region/CH 2-CH3 region/CH 2 region/CH 3 region is described as comprising a position/substitution "corresponding" to a reference position/substituent, equivalent positions/substitutions in the homologous heavy chain constant region/Fc region/CH 2-CH3 region/CH 2 region/CH 3 region are contemplated.

When the Fc region is described as comprising a particular position/substitution, that position/substitution may be present in one or both of the polypeptide chains that together form the Fc region.

Unless otherwise indicated, positions herein refer to positions of amino acid Sequences of the constant regions of human immunoglobulins numbered according to the EU numbering system, as in Kabat et al, Sequences of Proteins of Immunological Interest (Sequences of Proteins of Immunological Interest), 5 th edition, public health services, national institutes of health, Besserda, Maryland (1991). For example, the substitutions L242C and K334C in human IgG1 correspond to the amino acid sequences as set forth in SEQ ID NOs: 171 of the constant region of human IgG1, an L > C substitution at position 125, and a K > C substitution at position 217.

A homologous heavy chain constant region is a heavy chain constant region comprising an amino acid sequence that has at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the heavy chain constant region of human IgG1 (i.e., the amino acid sequence set forth in SEQ ID NO: 171). A homologous Fc region is an Fc region composed of a polypeptide comprising an amino acid sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the CH2-CH3 region of human IgG1 (i.e., the amino acid sequences shown in SEQ ID NOS: 174 and 175). The homologous CH2 region is a CH2 region comprising an amino acid sequence that has at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to one of human IgG1 (i.e., the amino acid sequence set forth in SEQ ID NO: 174). The homologous CH3 region is a CH3 region comprising an amino acid sequence that has at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to one of human IgG1 (i.e., the amino acid sequence set forth in SEQ ID NO: 175).

The corresponding position to that recognized in human IgG1 can be identified by sequence alignment, for example, using the sequence alignment software Clustalomega et al

J.2005，Bioinformatics 21，951-960)。

In some embodiments, the antigen binding molecules of the present invention comprise an Fc region comprising a modification to increase Fc-mediated functions. In some embodiments, the Fc region comprises a modification to increase ADCC effect. In some embodiments, the Fc region comprises a modification to increase ADCP action. In some embodiments, the Fc region comprises a modification to increase CDC effect. An antigen binding molecule comprising a modification of the Fc region to enhance Fc-mediated functions (e.g., ADCC, ADCP, CDC) induces a higher level of the relevant effector function than an antigen binding molecule comprising a corresponding unmodified Fc region.

In some embodiments, the antigen binding molecules of the present invention comprise an Fc region comprising a modification to increase affinity for one or more Fc receptors (e.g., Fc γ RIIa, Fc γ RIIIa). Modifications that increase affinity for Fc receptors may increase Fc-mediated effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the antigen binding molecules of the invention comprise an Fc region comprising a modification that reduces affinity for C1q, such modification desirably reducing Complement Dependent Cytotoxicity (CDC). In some embodiments, the antigen binding molecules of the present invention comprise an Fc region comprising a modification that increases hexamer formation. Fc region modifications that can increase the affinity of one or more Fc receptors, decrease the affinity of C1q, and/or increase hexamer formation are described, for example, in Saxena and Wu, Front Immunol. (2016)7:580, the entire contents of which are hereby incorporated by reference. In some embodiments, the antigen binding molecules of the present invention comprise an Fc region comprising CH2/CH3 comprising one or more of the substitutions shown in table 1 of Saxena and Wu, Front immunol. (2016)7: 580.

In some embodiments, the antigen binding molecules of the present invention comprise an Fc comprising a modification that increases binding to an Fc receptor. In some embodiments, the Fc region comprises a modification that increases binding to an fey receptor. In some embodiments, the Fc region comprises a modification that increases binding to one or more of Fc γ RI, Fc γ RIIa, Fc γ RIIb, Fc γ RIIc, Fc γ RIIIa, and Fc γ RIIIb. In some embodiments, the Fc region comprises a modification that increases binding to Fc γ RIIIa. In some embodiments, the Fc region comprises a modification that increases binding to Fc γ RIIa. In some embodiments, the Fc region comprises a modification that increases binding to Fc γ RIIb. In some embodiments, the Fc region comprises a modification that increases binding to FcRn. In some embodiments, the Fc region comprises a modification that increases binding to a complement protein. In some embodiments, the Fc region comprises a modification that increases or decreases binding to C1 q. In some embodiments, the Fc region comprises a modification that promotes hexamerization of the antigen binding molecule. In some embodiments, the Fc region comprises a modification that increases the half-life of the antigen binding molecule. In some embodiments, the Fc region comprises a modification that increases co-conjugation.

In the present specification, an "Fc γ receptor" may be from any species and includes isoforms, fragments, variants (including mutants) or homologues from any species. Similarly, "Fc γ RI", "Fc γ RIIa", "Fc γ RIIb", "Fc γ RIIc", "Fc γ RIIIa" and "Fc γ RIIIb" refer to Fc γ RI/Fc γ RIIa/Fc γ RIIb/Fc γ RIIc/Fc γ RIIIa/Fc γ RIIIb, respectively, from any species, including isoforms, fragments, variants (including mutants), or homologs from any species. Humans have six different classes of Fc γ receptors (mouse orthologs are shown in parentheses): fc γ RI (mFc γ RI), Fc γ RIIa (mFc γ RIII), Fc γ RIIb (mFc γ RIIb), Fc γ RIIc, Fc γ RIIIa (mFc γ RIV), and Fc γ RIIIb. Variant Fc γ receptors include, for example, the 158V and 158F polymorphs of human Fc γ RIIIa, and the 167H and 167R polymorphs of human Fc γ RIIa.

In some embodiments, the antigen binding molecules of the invention comprise an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region or comprising a CH2-CH3 region) one or more of the following (e.g., 1, 2, 3, 4, 5, 6, 7, or 8): c is at a position corresponding to bit 242; c is at a position corresponding to 334 bits; a is at a position corresponding to bit 236; d is at a position corresponding to bit 239; e at a position corresponding to bit 332; l is at a position corresponding to bit 330; k is at a position corresponding to 345 bits; g is at a position corresponding to bit 430.

In some embodiments, the antigen binding molecules of the invention comprise an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region or comprising a CH2-CH3 region) one or more of the following substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, or 8): L242C, K334C, G236A, S239D, I332E, a330L, E345K, and E430G.

In some embodiments, the antigen binding molecule comprises an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) a C at a position corresponding to position 242. In some embodiments, the Fc region comprises (e.g., comprises one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) a C at a position corresponding to position 334. In some embodiments, the Fc region comprises (e.g., comprises one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) a C at a position corresponding to position 242 and a C at a position corresponding to position 334.

In some embodiments, the antigen binding molecule comprises an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) an a at a position corresponding to position 236. In some embodiments, the Fc region comprises (e.g., comprises one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) a D at a position corresponding to position 239. In some embodiments, the Fc region comprises (e.g., comprises one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) an a at a position corresponding to position 236 and a D at a position corresponding to position 239.

In some embodiments, the antigen binding molecule comprises an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) an E at a position corresponding to position 332. In some embodiments, the Fc region comprises (e.g., comprises one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) an a at a position corresponding to position 236, a D at a position corresponding to position 239, and an E at a position corresponding to position 332.

In some embodiments, the antigen binding molecule comprises an Fc comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) an L at a position corresponding to position 330. In some embodiments, the Fc region comprises (e.g., comprises one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) an a at a position corresponding to position 236, a D at a position corresponding to position 239, an E at a position corresponding to position 332, and an L at a position corresponding to position 330.

In some embodiments, the antigen binding molecule comprises an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH3 region) a K at a position corresponding to position 345. In some embodiments, the Fc region comprises (e.g., comprises one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH3 region) a G at a position corresponding to position 430. In some embodiments, the Fc region comprises (e.g., comprises one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) a K at a position corresponding to position 345 and a G at a position corresponding to position 430.

In some embodiments, the antigen binding molecule comprises an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) a C at a position corresponding to position 242, a C at a position corresponding to position 334, an a at a position corresponding to position 236, and a D at a position corresponding to position 239.

In some embodiments, the antigen binding molecule comprises an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) a C at a position corresponding to position 242, a C at a position corresponding to position 334, an a at a position corresponding to position 236, a D at a position corresponding to position 239, and an E at a position corresponding to position 332.

In some embodiments, the antigen binding molecule comprises an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) a C at a position corresponding to position 242, a C at a position corresponding to position 334, an a at a position corresponding to position 236, a D at a position corresponding to position 239, an E at a position corresponding to position 332, and an L at a position corresponding to position 330.

In some embodiments, the antigen binding molecule comprises an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region) a C at a position corresponding to position 242, a C at a position corresponding to position 334, a K at a position corresponding to position 345, and a G at a position corresponding to position 430.

In some embodiments, the antigen binding molecule comprises an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) a L242C substitution (or equivalent substitution). In some embodiments, the Fc region comprises (e.g., comprises one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) a K334C substitution (or equivalent substitution). In some embodiments, the Fc region comprises (e.g., comprises one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) a L242C substitution (or equivalent substitution) and a K334C substitution (or equivalent substitution).

In some embodiments, the antigen binding molecule comprises an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) a G236A substitution (or equivalent substitution). In some embodiments, the Fc region comprises (e.g., comprises one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) a S239D substitution (or equivalent substitution). In some embodiments, the Fc region comprises (e.g., comprises one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) a G236A substitution (or equivalent substitution) and a S239D substitution (or equivalent substitution).

In some embodiments, the antigen binding molecule comprises an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) the I332E substitution (or equivalent substitution). In some embodiments, the Fc region comprises (e.g., comprises one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) a G236A substitution (or equivalent substitution), a S239D substitution (or equivalent substitution), and an I332E substitution (or equivalent substitution).

In some embodiments, the antigen binding molecule comprises an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) a330L substitution (or equivalent substitution). In some embodiments, the Fc region comprises (e.g., comprises one or more polypeptides comprising a heavy chain constant region, or comprising a CH2-CH3 region or a CH2 region) a G236A substitution (or equivalent substitution), a S239D substitution (or equivalent substitution), an I332E substitution (or equivalent substitution), and a330L substitution (or equivalent substitution).

In some embodiments, the antigen binding molecule comprises an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH3-CH3 region or a CH3 region) an E345K substitution (or equivalent substitution). In some embodiments, the Fc region comprises (e.g., comprises one or more polypeptides comprising a heavy chain constant region, or comprising a CH3-CH3 region or a CH3 region) an E430G substitution (or equivalent substitution). In some embodiments, the Fc region comprises (e.g., comprises one or more polypeptides comprising a heavy chain constant region, or comprising a CH3-CH3 region or a CH2 region) an E345K substitution (or equivalent substitution) and an E430G substitution (or equivalent substitution).

In some embodiments, the antigen binding molecule comprises an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH3-CH3 region or a CH2 region) a L242C substitution (or equivalent substitution), a K334C substitution (or equivalent substitution), a G236A substitution (or equivalent substitution), and a S239D substitution (or equivalent substitution).

In some embodiments, the antigen binding molecule comprises an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH3-CH3 region or a CH2 region) a L242C substitution (or equivalent substitution), a K334C substitution (or equivalent substitution), a G236A substitution (or equivalent substitution), a S239D substitution (or equivalent substitution), and an I332E substitution (or equivalent substitution).

In some embodiments, the antigen binding molecule comprises an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH3-CH3 region or a CH2 region) a L242C substitution (or equivalent substitution), a K334C substitution (or equivalent substitution), a G236A substitution (or equivalent substitution), a S239D substitution (or equivalent substitution), an I332E substitution (or equivalent substitution), and a330L substitution (or equivalent substitution).

In some embodiments, the antigen binding molecule comprises a Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH3-CH3 region) a L242C substitution (or equivalent substitution), a K334C substitution (or equivalent substitution), an E345K substitution (or equivalent substitution), and an E430G substitution (or equivalent substitution).

In some embodiments, the antigen binding molecule comprises an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH3-CH3 region) one or more of the following (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12): l at the position corresponding to position 243, P at the position corresponding to position 292, L at the position corresponding to position 300, I at the position corresponding to position 305 and L at the position corresponding to position 396; d at the position corresponding to 239 bits and E at the position corresponding to 332 bits; d corresponds to the position of 239 bits, E corresponds to the position of 332 bits and L corresponds to the position of 330 bits; a corresponds to position 298, A corresponds to position 333 and A corresponds to position 334; y corresponds to bit 234, Q corresponds to bit 235, W corresponds to bit 236, M corresponds to bit 239, D corresponds to bit 268, E corresponds to bit 270, and A corresponds to bit 298; e at the position corresponding to 270 bits, D at the position corresponding to 326 bits, M at the position corresponding to 330 bits and E at the position corresponding to 334 bits; a at the position corresponding to bit 236, D at the position corresponding to bit 239 and E at the position corresponding to bit 332; w corresponds to 326 bits and S corresponds to 333 bits; e at the position corresponding to 267 bits, F at the position corresponding to 268 bits and T at the position corresponding to 324 bits; r at position corresponding to 345 bits, G at position corresponding to 430 bits and Y at position corresponding to 440 bits; y corresponds to bit 252, T corresponds to bit 254 and E corresponds to bit 256; l at the position corresponding to 428 bits and S at the position corresponding to 434 bits.

In some embodiments, the antigen binding molecule comprises an Fc region comprising (e.g., comprising one or more polypeptides comprising a heavy chain constant region, or comprising a CH3-CH3 region) a combination of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) substitutions (or corresponding substitutions) of: F243L/R292P/Y300L/V305I/P396L; S239D/I332E; S239D/I332E/A330L; S298A/E333A/K334A; L234Y/L235Q/G236W/S239M/H268D/D270E/S298A; D270E/K326D/A330M/K334E; G236A/S239D/I332E; K326W/E333S; S267E/H268F/S324T; E345R/E430G/S440Y; M252Y/S254T/T256E; and M428L/N434S.

Polypeptides

The invention also provides polypeptide components of the antigen binding molecules. The polypeptide may be provided in isolated or substantially purified form.

The antigen binding molecule of the invention may be or may comprise a complex of polypeptides.

In the present specification, where a polypeptide comprises more than one domain or region, it will be appreciated that multiple domains/regions are preferably present in the same polypeptide chain. That is, a polypeptide comprising more than one domain or region is a fusion polypeptide comprising domains/regions.

In some embodiments, a polypeptide according to the invention comprises or consists of a VH as described herein. In some embodiments, a polypeptide according to the invention comprises or consists of a VL as described herein.

In some embodiments, the polypeptide further comprises one or more antibody heavy chain constant regions (CHs). In some embodiments, the polypeptide additionally comprises one or more antibody light chain constant regions (CL). In some embodiments, the polypeptide comprises a CH1, CH2 region, and/or CH3 region of an immunoglobulin (Ig).

In some embodiments, the polypeptide comprises one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments, the polypeptide comprises a CH1 region as described herein. In some embodiments, the polypeptide comprises a CH1-CH2 hinge region as described herein. In some embodiments, the polypeptide comprises a CH2 region as described herein. In some embodiments, the polypeptide comprises a CH3 region as described herein. In some embodiments, the polypeptide comprises a CH2-CH3 region as described herein.

In some embodiments, the polypeptide comprises a CH3 region, the CH3 region comprising any one of the following amino acid substitutions/combination of amino acid substitutions (e.g., as shown in table 1 of Ha et al, front. immnol (2016) 7: 394, incorporated by reference above): T366W; T366S, L368A and Y407V; T366W and S354C; T366S, L368A, Y407V and Y349C; S364H and F405A; Y349T and T394F; T350V, L351Y, F405A and Y407V; T350V, T366L, K392L and T394W; K360D, D399M and Y407A; E345R, Q347R, T366V and K409V; K409D and K392D; D399K and E356K; K360E and K409W; Q347R, D399V and F405T; K360E, K409W and Y349C; Q347R, D399V, F405T and S354C; K370E and K409W; and E357N, D399V and F405T.

In some embodiments, the CH2 and/or CH3 regions of a polypeptide comprise one or more amino acid substitutions for facilitating association of the polypeptide with another polypeptide comprising a CH2 and/or CH3 region.

In some embodiments, the polypeptide comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the polypeptide comprises a CL region described herein.

In some embodiments, the polypeptide of the invention comprises, from N-terminus to C-terminus, one of the following structures:

(i)VH

(ii)VL

(iii)VH-CH1

(iv)VL-CL

(v)VL-CH1

(vi)VH-CL

(vii)VH-CH1-CH2-CH3

(viii)VL-CL-CH2-CH3

(ix)VL-CH1-CH2-CH3

(x)VH-CL-CH2-CH3

the invention also provides antigen binding molecules comprised of the polypeptides of the invention. In some embodiments, the antigen binding molecules of the invention comprise one of the following combinations of polypeptides:

(A)VH+VL

(B)VH-CH1+VL-CL

(C)VL-CH1+VH-CL

(D)VH-CH1-CH2-CH3+VL-CL

(E)VH-CL-CH2-CH3+VL-CH1

(F)VL-CH1-CH2-CH3+VH-CL

(G)VL-CL-CH2-CH3+VH-CH1

(H)VH-CH1-CH2-CH3+VL-CL-CH2-CH3

(I)VH-CL-CH2-CH3+VL-CH1-CH2-CH3

in some embodiments, the antigen binding molecule comprises more than one of the combinations of polypeptides shown in (a) to (I) above. For example, with reference to (D) above, in some embodiments, the antigen binding molecule comprises two polypeptides comprising the structure VH-CH1-CH2-CH3, and two polypeptides comprising the structure VL-CL.

In some embodiments, the antigen binding molecules of the invention comprise one of the following combinations of polypeptides:

(J) VH (anti-HER 3) + VL (anti-HER 3)

(K) VH (anti-HER 3) -CH1+ VL (anti-HER 3) -CL

(L) VL (anti-HER 3) -CH1+ VH (anti-HER 3) -CL

(M) VH (anti-HER 3) -CH1-CH2-CH3+ VL (anti-HER 3) -CL

(N) VH (anti-HER 3) -CL-CH2-CH3+ VL (anti-HER 3) -CH1

(O) VL (anti-HER 3) -CH1-CH2-CH3+ VH (anti-HER 3) -CL

(P) VL (anti-HER 3) -CL-CH2-CH3+ VH (anti-HER 3) -CH1

(Q) VH (anti-HER 3) -CH1-CH2-CH3+ VL (anti-HER 3) -Cl-CH2-CH3

(R) VH (anti-HER 3) -CL-CH2-CH3+ VL (anti-HER 3) -CH1-CH2-CH3

Wherein: "VH (anti-HER 3)" refers to the VH of an antigen binding molecule capable of binding HER3 as described herein, as defined in one of (1) to (61); "VL (anti-HER 3)" refers to the VL of an antigen binding molecule capable of binding to HER3 as described herein, as defined in one of (62) to (119).

In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to an amino acid sequence set forth in one of SEQ ID NOs 187 to 223.

Linkers and additional sequences

In some embodiments, the antigen binding molecules and polypeptides of the invention comprise a hinge region. In some embodiments, a hinge region is provided between the CH1 region and the CH2 region. In some embodiments, a hinge region is provided between the CL region and the CH2 region. In some embodiments, the hinge region comprises or consists of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID No. 173.

In some embodiments, the antigen binding molecules and polypeptides of the invention comprise one or more linker sequences between the amino acid sequences. Linker sequences may be provided at one or both ends of one or more of the VH, VL, CH1-CH2 hinge region, CH2 region and CH3 region of the antigen binding molecule/polypeptide.

Linker sequences are known to the skilled worker and are described, for example, in Chen et al, Adv Drug Deliv Rev (2013)65(10): 1357-. In some embodiments, the linker sequence may be a flexible linker sequence. The flexible linker sequence allows relative movement of the amino acid sequences connected by the linker sequence. Flexible linkers are known to the skilled person, several of which are identified in Chen et al, Adv Drug Deliv Rev (2013)65(10): 1357-. Flexible linker sequences typically contain a high proportion of glycine and/or serine residues.

In some embodiments, the linker sequence comprises at least one glycine residue and/or at least one serine residue. In some embodiments, the linker sequence consists of glycine and serine residues. In some embodiments, the linker sequence is 1-2, 1-3, 1-4, 1-5, or 1-10 amino acids in length.

The antigen binding molecules and polypeptides of the invention may additionally comprise other amino acids or amino acid sequences. For example, the antigen binding molecules and polypeptides may comprise one or more amino acid sequences to facilitate expression, folding, transport, processing, purification, or detection of the antigen binding molecules/polypeptides. For example, the antigen binding molecule/polypeptide may optionally comprise a sequence encoding a His (e.g., 6Xhis), Myc, GST, MBP, FLAG, HA, E, or biotin tag at the N-or C-terminus of the antigen binding molecule/polypeptide. In some embodiments, the antigen binding molecule/polypeptide comprises a detectable moiety, such as a fluorescent, luminescent, immunodetectable, radioactive, chemical, nucleic acid, or enzymatic label.

The antigen binding molecules and polypeptides of the invention may additionally comprise a signal peptide (also referred to as a leader sequence or signal sequence). The signal peptide typically consists of a sequence of 5-30 hydrophobic amino acids, forming a single alpha helix. Secreted and cell surface expressed proteins typically comprise a signal peptide.

The signal peptide may be present at the N-terminus of the antigen binding molecule/polypeptide and may be present in the newly synthesized antigen binding molecule/polypeptide. The signal peptide provides for efficient transport and secretion of the antigen binding molecule/polypeptide. The signal peptide is typically removed by cleavage and is therefore not included in the mature antigen-binding molecule/polypeptide secreted from the cell expressing the antigen-binding molecule/polypeptide.

Signal peptides for a number of proteins are known and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information resources (Protein Information resources), Protein databases (Protein Data Bank), Ensembl and InterPro, and/or can be identified/predicted, for example using amino acid sequence analysis tools such as SignalP (Petersen et al 2011Nature Methods 8: 785-.

In some embodiments, the signal peptide of the antigen binding molecules/polypeptides of the invention comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of one of SEQ ID NOs 178 to 186.

Labels and conjugates

In some embodiments, the antigen binding molecules of the invention additionally comprise a detectable label.

In some embodiments, the antigen binding molecule comprises a detectable moiety, such as a fluorescent label, a phosphorescent label, a luminescent label, an immunologically detectable label (e.g., an epitope tag), a radioactive label, a chemical, nucleic acid, or enzymatic label. The antigen binding molecule may be covalently or non-covalently labeled with a detectable moiety.

Fluorescent labels include, for example, fluorescein, rhodamine, allophycocyanin, eosin and NDB, rare earth Green Fluorescent Proteins (GFP), such as europium (Eu), terbium (Tb) and samarium (Sm) chelates, tetramethylrhodamine, Texas Red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3 and Cy 5. The radioactive label comprises a radioactive isotope, such as iodine ¹²³ Iodine, iodine ¹²⁵ Iodine, iodine ¹²⁶ Iodine, iodine ¹³¹ Iodine, iodine ¹³³ Bromine ⁷⁷ Technetium ^99m And indium ¹¹¹ Indium, indium ^113m Gallium, gallium ⁶⁷ Gallium, gallium ⁶⁸ Ruthenium (II) and (III) ⁹⁵ Ruthenium (II) and (III) ⁹⁷ Ruthenium (II) and (III) ¹⁰³ Ruthenium (II) and (III) ¹⁰⁵ Mercury, mercury ²⁰⁷ Mercury, mercury ²⁰³ Rhenium ^99m Rhenium ¹⁰¹ Rhenium ¹⁰⁵ Scandium (III) ⁴⁷ Tellurium ^121m Tellurium ^122m Tellurium ^125m Thulium, thulium ¹⁶⁵ Thulium, thulium ¹⁶⁷ Thulium, thulium ¹⁶⁸ Copper, copper ⁶⁷ Fluorine ¹⁸ Yttrium, yttrium ⁹⁰ Palladium, palladium ¹⁰⁰ Bismuth, bismuth ²¹⁷ And antimony ²¹¹ . Luminescent labels include radioactive, chemiluminescent (e.g., acridinium ester, luminol, isoluminol), and bioluminescent labels. Immunodetectable labels include haptens, peptides/polypeptides, antibodies, receptors and ligands, such as biotin, avidin, streptavidin or digoxigenin. Nucleic acid markers include aptamers. Enzyme labels include, for example, peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase, and luciferase.

In some embodiments, the antigen binding molecules of the invention are coupled to a chemical moiety. The chemical moiety may be a moiety for providing a therapeutic effect. The chemical moiety may be a moiety for providing a therapeutic effect. Antibody-drug conjugates were administered in Parslow et al, biomedicines.2016, 9 months; 4(3) and 14 (general description). In some embodiments, the chemical moiety may be a drug moiety (e.g., a cytotoxic agent). In some embodiments, the drug moiety can be a chemotherapeutic agent. In some embodiments, the drug moiety is selected from calicheamicin, DM1, DM4, monomethyl auristatin e (mmae), monomethyl auristatin f (mmaf), SN-38, doxorubicin, duocarmycin (duocarmycin), D6.5, and PBD.

Specific exemplary embodiments of antigen binding molecules

In some embodiments, the antigen binding molecule comprises or consists of a polypeptide that:

(i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO. 187; and

(ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 188.

(i) two polypeptides comprising or consisting of an amino acid sequence having 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO: 189; and

(ii) Two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 190.

(i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO. 191; and

(ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 192.

(i) Two polypeptides comprising or consisting of an amino acid sequence having 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 193; and

(ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 195.

(i) two polypeptides comprising or consisting of an amino acid sequence having 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO: 194; and

(i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO: 196; and

(i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 197; and

(ii) Two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 199.

(i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 198; and

(i) Two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 200; and

(ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 201.

(i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 202; and

(ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO. 203.

(i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 204; and

(ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 205.

(i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO. 206; and

(ii) Two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 207.

(i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 208; and

(ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 209.

(i) Two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 210; and

(ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 211.

(i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 212; and

(ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 213.

(i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 214; and

(ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 215.

(i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 216; and

(ii) Two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 217.

(i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 218; and

(ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO: 219.

(i) Two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID No. 220; and

(ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 221.

(i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 222; and

(ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 223.

(i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 225; and

(ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 207.

(i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 226; and

(i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 227; and

(ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 217.

(i) Two polypeptides comprising or consisting of an amino acid sequence having 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence depicted in SEQ ID NO 228; and

Functional Properties of antigen binding molecules

The antigen binding molecules described herein are characterized with reference to certain functional properties. In some embodiments, the antigen binding molecules described herein can have one or more of the following properties:

binds to HER3 (e.g. human, mouse, rat or cynomolgus HER 3);

does not bind to EGFR and/or HER 2;

binding to a cell expressing HER 3;

binding to subdomain II of the extracellular region of HER 3;

binds to HER3 when HER3 is in open and closed conformations;

binds to HER3 independently of NRG;

Does not compete with MM-121 and/or LJM-716 for binding to HER 3;

does not compete with M-05-74 and/or M-08-11 for binding to HER 3;

inhibit the interaction between HER3 and HER3 interaction partners (e.g., HER3, HER2, EGFR, HER4, HGFR, IGF1R, and/or cMet);

inhibit HER 3-mediated signaling;

inhibiting proliferation of HER 3-expressing cells (e.g., in response to NRG stimulation);

inhibition of PI3K/AKT/mTOR and/or MAPK signaling (e.g., in response to NRG stimulation) by HER 3-expressing cells;

binding to an activated Fc γ receptor (e.g., Fc γ RIIIa);

increased binding to an activating Fc γ receptor;

increased binding to an activating Fc gamma receptor as compared to an equivalent antigen binding molecule having an Fc region consisting of CH2-CH3 having the amino acid sequence shown in SEQ ID NO 174-175;

reduced binding to an inhibitory Fc γ receptor as compared to an equivalent antigen-binding molecule having an Fc region consisting of CH2-CH3 having the amino acid sequence shown in SEQ ID NO: 174-175;

increased binding to an activating Fc γ receptor relative to an inhibiting Fc γ receptor as compared to an equivalent antigen binding molecule having an Fc region consisting of CH2-CH3 having the amino acid sequence set forth in SEQ ID NO 174-175;

increased or decreased binding to a complement protein (e.g., C1q) as compared to an equivalent antigen binding molecule having an Fc region consisting of CH2-CH3 of the amino acid sequence set forth in SEQ ID NO: 174-175;

An increase in hexamers compared to an equivalent antigen binding molecule having an Fc region consisting of CH2-CH3 of the amino acid sequence shown in SEQ ID NO: 174-175;

an increased ADCC activity compared to an equivalent antigen binding molecule having an Fc region consisting of CH2-CH3 of the amino acid sequence shown in SEQ ID NO: 174-175;

increased ADCP activity compared to an equivalent antigen binding molecule having an Fc region consisting of CH2-CH3 of the amino acid sequence shown in SEQ ID NO: 174-175;

increased or decreased CDC activity as compared to an equivalent antigen binding molecule having an Fc region consisting of CH2-CH3 of the amino acid sequence shown in SEQ ID NO: 174-175;

and a polypeptide having a sequence defined by SEQ ID NO:174-175 from CH2-CH3 of the amino acid sequence, similar or increased thermostability;

increased killing of HER3 expressing cells;

reducing the number/proportion of cells expressing HER 3; and

inhibiting the development and/or progression of cancer in vivo.

The antigen binding molecules described herein preferably exhibit specific binding to HER 3. As used herein, "specific binding" refers to binding that is selective for an antigen and can be distinguished from non-specific binding to a non-target antigen. An antigen binding molecule that specifically binds to a target molecule preferably binds with greater affinity and/or for a longer duration of time to the target than to other non-target molecules.

The ability of a given polypeptide to specifically bind to a given molecule can be determined by assays according to Methods known in the art, e.g., by ELISA, surface plasmon resonance (SPR; see, e.g., Hearty et al, Methods Mol Biol (2012)907:411-442), biolayer interferometry (see, e.g., Lad et al, (2015) J Biomol Screen 20(4):498-507), flow cytometry or by radiolabeled antigen binding assay (RIA), enzyme-linked immunosorbent assay. By this analysis, the binding to a given molecule can be measured and quantified. In some embodiments, binding may be a response detected in a given assay.

In some embodiments, the degree of binding of the antigen binding molecule to the non-target molecule is less than about 10% of the degree of binding of the antibody to the target molecule, as determined by ELISA, SPR, biolayer interferometry, or RIA. Alternatively, binding specificity may be reflected in binding affinity, where the antigen binding molecule binds to the dissociation constant (K) _D ) K over antigen binding molecule to non-target molecule _D At least 0.1 orders of magnitude greater (i.e., 0.1 x 10) ⁿ Where n is an integer representing an order of magnitude). This may optionally be at least one of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0.

In some embodiments, the antigen binding molecule exhibits binding to human HER3, mouse HER3, rat HER3, and/or cynomolgus monkey (cynomolgus monkey) HER 3. That is, in some embodiments, the antigen binding molecule is cross-reactive to human HER3, mouse HER3, rat HER3, and/or cynomolgus monkey HER 3. In some embodiments, the antigen binding molecules of the invention exhibit cross-reactivity with non-human primate HER 3. Cross-reactivity with HER3 in model species allows the exploration of the effectiveness of homologous models in vivo, independent of surrogate molecules.

In some embodiments, the antigen binding molecule binds to human HER3, mouse HER3, rat HER3, and/or cynomolgus monkey HER 3; and does not bind to HER2 and/or EGFR (e.g., human HER2 and/or human EGFR).

In some embodiments, the antigen binding molecule does not exhibit specific binding to EGFR (e.g., human EGFR). In some embodiments, the antigen binding molecule does not exhibit specific binding to HER2 (e.g., human HER 2). In some embodiments, the antigen binding molecule does not exhibit specific binding to (i.e., does not cross-react with) an EGFR protein family member other than HER 3. In some embodiments, the antigen binding molecule does not exhibit specific binding to EGFR, HER2, and/or HER 4.

In some embodiments, the antigen binding molecules of the invention bind to K of HER3 (e.g., human HER3) _D Less than 10 μ M, preferably less than or equal to 5 μ M, less than or equal to 2 μ M, less than or equal to 1 μ M, less than or equal to 500nM, less than or equal to 400nM, less than or equal to 300nM, less than or equal to 200nM, less than or equal to 100nM, less than or equal to 90nM, less than or equal to 85nM, less than or equal to 80nM, less than or equal to 75nM, less than or equal to 70nM, less than or equal to 65nM, less than or equal to 60nM, less than or equal to 55nM, less than or equal to 50nM, less than or equal to 45nM, less than or equal to 40nM, less than or equal to 35nM, less than or equal to 30nM, less than or equal to 25nM, less than or equal to 20nM, less than or equal to 15nM, less than or equal to 12.5, less than or equal to 10nM, less than or equal to 9, less than or equal to 7, less than or equal to 6, less than or equal to 5, less than or equal to 4nM, less than or equal to 3, less than or equal to 200pM 200nM, less than or equal to 10pM 200nM, less than or equal to 10 pM3, less than or equal to 200pM 60, less than or equal to 200pM, less than or equal to 200nM, less than or equal to 200pM 200nM, less than or equal to 200nM, less than or equal to 10nM, less than or equal to 200pM, less than or equal to 200nM, less than or equal to 200pM 200nM, less than or equal to 10nM, less than or equal to 200nM, less than or equal to 200pM 200, less than or equal to 200 pM3, less than or equal to 200nM, less than or equal to 200 pM3, less than or equal to 200 pM10 nM, less than or equal to 200 pM10 nM, less than or equal to 200 pM3, less than or equal to 200pM, Less than or equal to one of 1 pM.

The antigen binding molecules of the invention can bind to a specific region of interest of HER 3. The antigen binding region of the antigen binding molecule according to the invention may bind to a linear epitope of HER3, which linear epitope of HER3 consists of a contiguous sequence of amino acids (i.e. the amino acid primary sequence). In some embodiments, the antigen binding molecule may bind to a conformational epitope of HER3 that consists of amino acids that are not contiguous in the amino acid sequence.

In some embodiments, the antigen binding molecule binds to the region of HER3 shown in SEQ ID No. 229. In some embodiments, the antigen binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID No. 229. In some embodiments, the antigen binding molecule binds to the region of HER3 shown in SEQ ID NOs 230 and 231. In some embodiments, the antigen binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID NOs 230 and 231. In some embodiments, the antigen binding molecule binds to the region of HER3 shown in SEQ ID NO 230. In some embodiments, the antigen binding molecule contacts one or more amino acid residues of the region of HER3 as set forth in SEQ ID No. 230. In some embodiments, the antigen binding molecule binds to the region of HER3 shown in SEQ ID No. 231. In some embodiments, the antigen binding molecule contacts one or more amino acid residues of the region of HER3 as set forth in SEQ ID No. 231. In some embodiments, the antigen binding molecule binds to the region of HER3 shown in SEQ ID NO 23. In some embodiments, the antigen binding molecule contacts one or more amino acid residues of the HER3 region shown in SEQ ID No. 23. In some embodiments, the antigen binding molecule binds to the region of HER3 shown in SEQ ID NO 21. In some embodiments, the antigen binding molecule contacts one or more amino acid residues of the HER3 region shown in SEQ ID No. 21. In some embodiments, the antigen binding molecule binds to the HER3 region shown in SEQ ID NO 19. In some embodiments, the antigen binding molecule contacts one or more amino acid residues of the HER3 region shown in SEQ ID No. 19. In some embodiments, the antigen binding molecule binds to the HER3 region shown in SEQ ID NO. 22. In some embodiments, the antigen binding molecule contacts one or more amino acid residues of the HER3 region shown in SEQ ID No. 22.

In some embodiments, the antigen binding molecules of the invention are capable of binding to a polypeptide comprising or consisting of an amino acid sequence set forth in one of

SEQ ID NOs

1, 3, 4, 6, or 8. In some embodiments, the antigen binding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO. 9. In some embodiments, the antigen binding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO. 16. In some embodiments, the antigen binding molecule is capable of binding to a peptide/polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID No. 229. In some embodiments, the antigen binding molecule is capable of binding to a peptide/polypeptide comprising or consisting of the amino acid sequences shown in SEQ ID NOs 230 and 231. In some embodiments, the antigen binding molecule is capable of binding to a peptide/polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 230. In some embodiments, the antigen binding molecule is capable of binding to a peptide/polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID No. 231. In some embodiments, the antigen binding molecule is capable of binding to a peptide/polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the antigen binding molecule is capable of binding to a peptide/polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID No. 21. In some embodiments, the antigen binding molecule is capable of binding to a peptide/polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID No. 19. In some embodiments, the antigen binding molecule is capable of binding to a peptide/polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID No. 22.

In some embodiments, the antigen binding molecule does not bind to the region of HER3 corresponding to positions 260 to 279 of SEQ ID No. 1. In some embodiments, the antigen binding molecule does not contact amino acid residues corresponding to the region of HER3 from positions 260 to 279 of SEQ ID No. 1. In some embodiments, the antigen binding molecule does not bind to the HER3 region shown in SEQ ID NO. 23. In some embodiments, the antigen binding molecule does not contact the amino acid residues of the region of HER3 as set forth in SEQ ID NO. 23. In some embodiments, the antigen binding molecule is not capable of binding to a peptide consisting of an amino acid sequence corresponding to positions 260 to 279 of the amino acid sequence depicted in SEQ ID NO 1. In some embodiments, the antigen binding molecule is not capable of binding to a peptide consisting of the amino acid sequence set forth in SEQ ID NO. 23.

As used herein, "peptide" refers to a chain of two or more amino acid monomers linked by peptide bonds. The length of the peptide is typically in the range of about 2 to 50 amino acids. A "polypeptide" is a polymer chain of two or more peptides. Polypeptides are typically greater than about 50 amino acids in length.

The ability of an antigen binding molecule to bind to a given peptide/polypeptide can be analyzed by methods well known to those skilled in the art, including by ELISA, immunoblotting (e.g., western blotting), immunoprecipitation, surface plasmon resonance, and biolayer interferometry.

Binding of ligand to HER3 promotes conformational changes that change HER3 into a homo-or heterodimer, thereby activating downstream pathways. HER3 exhibits "closed" and "open" conformations. Closed conformation means that HER3 is in a constrained conformation and is not available for receptor homodimerization or heterodimerization. By open conformation is meant HER3 in an extended conformation useful for receptor homodimerization or heterodimerization.

In some embodiments, the antigen binding molecule is capable of binding HER3 when HER3 is in an open conformation. In some embodiments, the antigen binding molecule is capable of binding HER3 when HER3 is in a closed conformation. In some embodiments, the antigen binding molecule is capable of binding to HER3 when HER3 is in an open and/or closed conformation. In some embodiments, the antigen binding molecule is capable of binding to HER3 extracellular domain when HER3 is in an open and/or closed conformation. In some embodiments, the antigen binding molecule is capable of binding to HER3 dimeric arm when HER3 is in an open and/or closed conformation. Binding to the dimeric arm enables the antigen binding molecule to prevent interaction between interacting partners of HER3 and HER3, such as described herein.

In some embodiments, the antigen binding molecule is capable of binding HER3 in the presence and/or absence of HER3 ligand. In some embodiments, the antigen binding molecule is capable of binding HER3 independently of the ligand of HER 3. In some embodiments, the ligand is NRG, NRG-1 and/or NRG-2. HER3 is activated by binding of a ligand to its extracellular domain, promoting a conformational change that renders HER3 homo-or heterodimeric. Binding of the antigen binding molecule to HER3 independent of ligand binding allows the antigen binding molecule to inhibit the action of HER3 in both the ligand-deficient and ligand-present conformational states. In some embodiments, the antigen binding molecule does not compete with the ligand for binding to HER 3. In some embodiments, the antigen binding molecule does not bind HER3 at the ligand binding site.

In some embodiments, the antigen binding molecule binds similarly well to HER3 in the presence or absence of ligand for HER3 (i.e., whether HER3 is provided in ligand bound or unbound form).

In some embodiments, the affinity of the antigen binding molecule to bind HER3 in the presence of HER3 ligand is similar to the affinity of the antigen binding molecule to bind HER3 in the absence of HER3 ligand. Example 8.10 and figures 78A and 78B of the present disclosure demonstrate that 10D1F binds human HER3 with sub-picomolar affinity when HER3 is provided in NRG1 bound form, and in the absence of NRG 1.

Herein, a binding affinity that is "similar" to a reference binding affinity refers to a binding affinity that is within 50% of the reference binding affinity determined under comparable conditions, e.g., within one of 40%, 45%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the reference binding affinity.

In some embodiments, the antigen binding molecule binds to K of HER3 in the presence of a ligand of HER3 (e.g., NRG1 or NRG2) _D Is K of an antigen binding molecule that binds HER3 in the absence of a ligand _D Within 50% (as measured under comparable conditions), e.g. within one of 40%, 45%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

In some embodiments, the antigen binding molecule binds to K of HER3 in the presence of a ligand of HER3 (e.g., NRG1 or NRG2) _on Is K of an antigen binding molecule that binds HER3 in the absence of a ligand _on (as measured under comparable conditions) within 50%, for example within one of 40%, 45%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

In some implementationsIn this way, the antigen binding molecule binds to K of HER3 in the presence of a ligand of HER3 (e.g. NRG1 or NRG2) _off Is K of an antigen binding molecule that binds HER3 in the absence of a ligand _off (as measured under comparable conditions) within 50%, for example within one of 40%, 45%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

In certain embodiments, the antigen binding molecule is capable of binding to the same region of HER3, or an overlapping region of HER3, in combination with HER3 comprising an antibody comprising VH and VL sequences selected from any one of the following clones: 10D1, 10D1_ c75, 10D1_ c76, 10D1_ c77, 10D1_ c78v1, 10D1_ c78v2, 10D1_11B, 10D1_ c85v1, 10D1_ c85v2, 10D1_ c85o1, 10D1_ c85o2, 10D1_ c87, 10D1_ c89, 10D1_ c90, 10D1_ c91, 10D1_ c92, 10D1_ c92, 10D1_ c93, 10A6, 4-35-B2 or 4-35-B4. In certain embodiments, the antigen binding molecule is capable of binding to the same region of HER3 or to an overlapping region of HER3, to a region of HER3 to which an antibody binds, said antibody comprising VH and VL sequences selected from any one of the following clones: 10D1_ c89, 10D1_ c90, or 10D1_ c 91. In some embodiments, the antigen binding molecule is capable of binding to the same region of HER3 or an overlapping region of HER3, the region of HER3 bound by an antibody comprising the VH and VL sequences of clone 10D1_ c 89.

The region of the peptide/polypeptide to which the antibody binds can be determined by the skilled artisan using a variety of methods well known in the art, including X-ray co-crystal analysis of antibody-antigen complexes, peptide scanning, mutagenesis mapping, mass spectrometry hydrogen-deuterium exchange analysis, phage display, competition ELISA and proteolysis-based "protection" methods. Such methods are described, for example: gershoni et al, BioDrugs, 2007, 21 (3): 145-156, which is incorporated herein by reference in its entirety. Such methods can also be used to determine whether an antigen binding molecule is capable of binding to a protein of a different conformation.

Antibodies that do not bind to HER3 in the same region of HER3 or in the overlapping region of HER3 in antibodies comprising the VH and VL sequences of anti-HER 3 antibody clone MM-121 (described, for example, in Schoeberl et al, Sci.Signal. (2009)2 (77): ra 31) and/or LJM-716 (described, for example, in Garner et al, Cancer Res (2013) 73: 6024-6035). In some embodiments, the antigen binding molecules of the invention do not compete with antibodies comprising the VH and VL sequences of anti-HER 3 antibody clone MM-121 and/or LJM-716 for binding to HER3, e.g., as determined by SPR analysis.

In some embodiments, when HER3 is expressed on the surface of a cell (i.e. at or at the cell membrane), the antigen binding molecules of the invention bind to HER3 (i.e. extracellular antigen binding molecules) in a region accessible to the antigen binding molecule. In some embodiments, the antigen binding molecule is capable of binding to HER3 expressed on the cell surface of a HER 3-expressing cell. In some embodiments, the antigen binding molecule is capable of binding to a cell that expresses HER3 (e.g., a HER3+ cell, such as a HER3+ cancer cell).

The ability of an antigen binding molecule to bind to a given cell type can be analyzed by contacting the cell with the antigen binding molecule and detecting the antigen binding molecule bound to the cell, e.g., after a washing step, removing unbound antigen binding molecule. The ability of the antigen binding molecules to bind to cells expressing immune cell surface molecules and/or cells expressing cancer cell antigens can be analyzed by methods such as flow cytometry and immunofluorescence microscopy.

The antigen binding molecule of the invention may be an antagonist of HER 3. In some embodiments, the antigen binding molecule is capable of inhibiting a function or process (e.g., interaction, signaling, or other activity) mediated by a binding partner of HER3 and/or HER3 (e.g., HER3 (i.e., in the case of homodimerization), HER2, EGFR, HER4, HGFR, IGF1R, and/or cMet). Herein, "inhibit" refers to a decrease, or attenuation relative to a control condition.

In some embodiments, the antigen binding molecules of the invention are capable of inhibiting the interaction between HER3 and HER3 interacting partners. The interacting partner of HER3 may be expressed by the same cell as HER 3. The interaction partner or HER3 may be expressed at the cell surface (i.e. in or at the cell membrane). In some embodiments, the interaction partner of HER3 may be a member of the EGFR protein family, e.g., HER3, HER2, EGFR, HER4, HGFR, IGF1R, and/or cMet. In some embodiments, the interaction partner of HER3 may be IGF1R and/or cMet. The interaction between HER3 and HER3 interacting partners may result in the formation of a polypeptide complex. The interaction between HER3 and HER3 interacting partners to form a polypeptide complex may be referred to as multimerization. In the case of multimerization between polypeptide monomers, multimerization may be referred to as dimerization.

In some embodiments, the antigen binding molecule is capable of inhibiting the interaction between HER3 monomers. In some embodiments, the antigen binding molecule is capable of inhibiting the interaction between HER3 and HER 2. In some embodiments, the antigen binding molecule is capable of inhibiting the interaction between HER3 and EGFR. In some embodiments, the antigen binding molecule is capable of inhibiting the interaction between HER3 and HER 4. In some embodiments, the antigen binding molecule is capable of inhibiting the interaction between HER3 and HGFR. In some embodiments, the antigen binding molecule is capable of inhibiting the interaction between HER3 and IGF 1R. In some embodiments, the antigen binding molecule is capable of inhibiting the interaction between HER3 and cMet.

Inhibition of the interaction may be achieved by binding of the antigen binding molecule to the region of HER3 required for the interaction between the interacting partners of HER3 and HER3 (e.g. the dimeric ring of HER3 as shown in SEQ ID NO: 19). In some embodiments, the antigen binding molecule contacts one or more residues of HER3 necessary for the interaction between the interaction partners of HER3 and HER 3; in this way, the antigen binding molecule renders this region unusable, thereby inhibiting the interaction. In some embodiments, the antigen binding molecule binds to HER3 in a manner that inhibits/prevents the interaction between HER3 and the HER3 interacting partner. In some embodiments, the antigen binding molecule inhibits/prevents the entry of the interaction partner of HER3 into the region of HER3 required for the interaction between the interaction partner of HER3 and HER 3; this can be achieved without the antigen binding molecule contacting the region of HER3 required for the interaction between HER3 and the interaction partner of HER3, for example, by sterically inhibiting the entry of the interaction partner of HER3 into the region of HER3 required for the interaction between HER3 and the interaction partner.

In some embodiments, the antigen binding molecule is capable of inhibiting homodimerization of HER3 monomer. In some embodiments, the antigen binding molecule is capable of inhibiting dimerization between HER3 and HER 2. In some embodiments, the antigen binding molecule is capable of inhibiting dimerization between HER3 and EGFR. In some embodiments, the antigen binding molecule is capable of inhibiting dimerization between HER3 and HER 4. In some embodiments, the antigen binding molecule is capable of inhibiting dimerization between HER3 and HGFR. In some embodiments, the antigen binding molecule is capable of inhibiting dimerization between HER3 and IGF 1R. In some embodiments, the antigen binding molecule is capable of inhibiting dimerization between HER3 and cMet.

The ability of an antigen binding molecule to inhibit the interaction between two factors can be determined, for example, by assaying the interaction after the antibody/fragment is present or incubated with one or both of the interacting partners. Assays for determining whether a given antigen binding molecule is capable of inhibiting the interaction between two interacting partners include competitive ELISA assays and SPR assays. In some embodiments, the antigen binding molecule is a competitive inhibitor of the interaction between HER3 and an interacting partner of HER 3.

In some embodiments, an antigen binding molecule of the invention is capable of inhibiting the interaction between an interacting partner of HER3 and HER3 (e.g., HER3, HER2, EGFR, HER4, HGFR, IGF1R and/or cMet) to a level of interaction between an interacting partner of HER3 and HER3 that is at least less than 1 fold, e.g., less than 0.99 fold, less than 0.95 fold, less than 0.9 fold, less than 0.85 fold, less than 0.8 fold, less than 0.75 fold, less than 0.7 fold, less than 0.65 fold, less than 0.6 fold, less than 0.55 fold, less than 0.5 fold, less than 0.45 fold, less than 0.4 fold, less than 0.35 fold, less than 0.3 fold, less than 0.25 fold, less than 0.05 fold, or less than 0.15 fold in the absence of an antigen binding molecule in a suitable assay.

The ability of an antigen binding molecule to inhibit the interaction between interacting partners can also be determined by assaying the downstream functional consequences of such an interaction. For example, downstream functional consequences of the interaction between HER3 and an interacting partner of HER3 include PI3K/AKT/mTOR and/or MAPK signaling. For example, the ability of an antigen binding molecule to inhibit the interaction of HER3 with an interaction partner of HER3 can be determined by analyzing PI3K/AKT/mTOR and/or MAPK signaling after treatment with an NRG molecule in the presence of antigen binding. PI3K/AKT/mTOR and/or MAPK signaling can be detected and quantified, for example, by using antibodies capable of detecting phosphorylated members of the signaling pathway.

The ability of an antigen binding molecule to inhibit the HER3 and HER3 interaction partner can also be determined by analyzing the proliferation of HER3 expressing cells after treatment with NRG in the presence of the antigen binding molecule. Cell proliferation can be measured, for example, by detecting changes in cell number over time, or by in vitro assays ³ Incorporation of H-thymidine or by CFSE dilution assay, e.g. as measured in Fulcher and Wong, immunocytologic organisms (1999)77 (6): 559-564, herein incorporated by reference in their entirety.

In some embodiments, the antigen binding molecules of the present invention are capable of inhibiting proliferation of cells having a mutation to BRAF V600, said cells comprising a BRAF V600E or V600K mutation (see example 10).

In some embodiments, the antigen binding molecule inhibits HER 3-mediated signaling. HER 3-mediated signal transduction may be assayed, for example, by using detection of HER 3-mediated signal-related factors, such as cellular proliferation, and/or phosphorylation of one or more signal transduction molecules of the PI3K/AKT/mTOR and/or MAPK signal transduction pathways.

In some embodiments, the antigen binding molecules of the invention are capable of inhibiting PI3K/AKT/mTOR and/or MAPK signaling by a cell expressing HER 3. The level of PI3K/AKT/mTOR and/or MAPK signaling can be analyzed by detecting and quantifying the level of phosphorylation of one or more components of the PI3K/AKT/mTOR and/or MAPK pathway. E.g. after NRG stimulation (see example 4.3).

In some embodiments, the antigen binding molecules of the invention are capable of inhibiting proliferation of a cell expressing HER3, e.g., in response to stimulation by NRG. In some embodiments, an antigen binding molecule of the invention is capable of inhibiting the proliferation of a cell expressing HER3 by at least less than 1 fold, e.g., less than or equal to 0.99 fold, less than or equal to 0.95 fold, less than or equal to 0.9 fold, less than or equal to 0.85 fold, less than or equal to 0.8 fold, less than or equal to 0.75 fold, less than or equal to 0.7 fold, less than or equal to 0.65 fold, less than or equal to 0.6 fold, less than or equal to 0.55 fold, less than or equal to 0.5 fold, less than or equal to 0.45 fold, less than or equal to 0.4 fold, less than or equal to 0.35 fold, less than or equal to 0.3 fold, less than or equal to 0.25 fold, less than or equal to 0.2 fold, less than or equal to 0.15 fold, less than or equal to 0.1 fold, less than or equal to 0.05 fold of a cell expressing HER3 in the absence of the antigen binding molecule.

In some embodiments, an antigen binding molecule of the invention is capable of inhibiting PI3K/AKT/mTOR and/or MAPK signaling in a suitable assay by a cell expressing HER3 at least less than 1 fold, e.g., less than or equal to 0.99 fold, less than or equal to 0.95 fold, less than or equal to 0.9 fold, less than or equal to 0.85 fold, less than or equal to 0.8 fold, less than or equal to 0.75 fold, less than or equal to 0.7 fold, less than or equal to 0.65 fold, less than or equal to 0.6 fold, less than or equal to 0.55 fold, less than or equal to 0.5 fold, less than or equal to 0.45 fold, less than or equal to 0.4 fold, less than or equal to 0.35 fold, less than or equal to 0.3 fold, less than or equal to 0.25 fold, less than or equal to 0.2 fold, less than or equal to 0.15 fold, less than or equal to 0.1 fold, less than or equal to 0.05 fold, less than or equal to 0.01 fold of a cell expressing HER3 in the absence of antigen binding molecule.

HER3 mediated signaling can be studied in vitro, e.g. as described in example 8.9, or in vivo, e.g. as described in example 11.

ADCC activity can be measured, for example, by the methods described in Yamashita et al, scientific report (2016) 6: 19772 (incorporated herein by reference in its entirety), or by a method such as that described in Jedema et al, blood (2004) 103: 2677-82 (incorporated herein by reference in their entirety) ⁵¹ Cr release test analysis. ADCC activity can also be assayed using the Pierce LDH cytotoxicity assay kit (as described in example 5 herein) according to the manufacturer's instructions.

ADCP can be analyzed, for example, according to the methods described in Kamen et al, J Immunol (2017)198(1 suppl) 157.17 (incorporated herein by reference in its entirety).

The ability to induce CDC may be determined, for example, by using a C1q binding assay, such as Schlothauer et al, protein engineering, design and screening (2016), 29 (10): 457-466 (which is incorporated herein by reference in its entirety).

The thermal stability of antigen binding molecules can be analyzed by methods well known to those skilled in the art, including, for example, differential scanning photometry and Differential Scanning Calorimetry (DSC) as described in He et al, J Pharm Sci. (2010), which is incorporated herein by reference in its entirety. The thermal stability may be measured by the melting temperature (T) _m ) The development temperature or decomposition temperature (e.g., in degrees c or F °).

) The affinity of (a) is a polypeptide comprising SEQ ID NO: 174-175 the equivalent antigen binding molecule of the Fc region consisting of CH2-CH3 of the amino acid sequence shown binds greater than 1-fold, e.g., greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15-fold, or greater than 20-fold, with an affinity for activating Fc γ receptors. In some embodiments, an antigen binding molecule comprising an Fc region described herein binds to K of an activating fey receptor _D Is a polypeptide comprising SEQ ID NO: 174-175 equivalent antigen binding molecules to the Fc region consisting of CH2-CH3 of the amino acid sequence shown in SEQ ID NO: 174 bind to K activating the Fc gamma receptor _D Less than 1 times, e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06 times, or less than 0.05 times.

In some embodiments, the antigen binding molecule comprising an Fc region as described herein binds to K of an activating Fc γ receptor (e.g., hFc γ RIIa167H, hFc γ RIIa167R), hFc γ RIIIa (e.g., hFc γ RIIIa158V, hFc γ RIIIa158F), mfcγ RIV, mfcγ RIII) _D Less than or equal to 1000nM, preferably less than or equal to 500nM, less than or equal to 100nM, less than or equal to 75nM, less than or equal to 50nM, less than or equal to 40nM, less than or equal to 30nM, less than or equal to 20nM, less than or equal to 15nM, less than or equal to 12.5nM, less than or equal to 10nM, less than or equal to 9nM, less than or equal to 8nM, less than or equal to 7nM, less than or equal to 6nM, less than or equal to 5nM, less than or equal to 4nM, less than or equal to 3nM, less than or equal to 2nM or less than or equal to 1 nM.

In some embodiments, an antigen binding molecule comprising an Fc region as described herein binds FcRn (e.g., hFcRn, mFcRn) with affinity to FcRn comprising SEQ ID NO: 174-175 the equivalent antigen binding molecule of the Fc region consisting of CH2-CH3 of the amino acid sequence shown in seq id No. binds FcRn with greater than 1-fold, e.g., greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15-fold, or greater than 20-fold affinity. In some embodiments, an Fc region as described herein is includedThe antigen binding molecule of (a) binds to K of FcRn _D Is a polypeptide comprising SEQ ID NO: 174-175 equivalent antigen binding molecules to the Fc region consisting of CH2-CH3 of the amino acid sequence depicted in FcRn bind to K of FcRn _D Less than 1 times, e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06 times, or less than 0.05 times.

In some embodiments, an antigen binding molecule comprising an Fc region described herein binds to K of FcRn (e.g., hFcRn, mFcRn) _D Less than or equal to 1000nM, preferably less than or equal to 500nM, less than or equal to 100nM, less than or equal to 75nM, less than or equal to 50nM, less than or equal to 40nM, less than or equal to 30nM, less than or equal to 20nM, less than or equal to 15nM, less than or equal to 12.5nM, less than or equal to 10nM, less than or equal to 9nM, less than or equal to 8nM, less than or equal to 7nM, less than or equal to 6nM, less than or equal to 5nM, less than or equal to 4nM, less than or equal to 3nM, less than or equal to 2nM or less than or equal to 1 nM.

In some embodiments, the affinity of an antigen binding molecule comprising an Fc region described herein for binding to an inhibitory Fc γ receptor (e.g., hFc γ RIIb, mFc γ RIIb) is a peptide comprising SEQ ID NO: 174-175 the equivalent antigen binding molecule of the Fc region consisting of CH2-CH3 of the amino acid sequence shown binds less than 1-fold, e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2-fold, or less than 0.1-fold, of the inhibitory Fc γ receptor. In some embodiments, a K comprising an Fc region that binds to an antigen binding molecule of an inhibitory fey receptor as described herein _{D is} Comprises the amino acid sequence shown in SEQ ID NO: 174-175 of the amino acid sequence CH2-CH3 inhibits K of Fc gamma receptor _D Greater than 1 times, such as greater than 2, 3, 4, 5, 6, 7, 8, 9 times, or greater than 10 times.

In some embodiments, an antigen binding molecule comprising an Fc region as described herein binds to K of an inhibitory Fc γ receptor (e.g., hfcyriibibffcyriib) _D Greater than or equal to 1nM, preferably greater than or equal to one of 5nM, greater than or equal to 10nM, greater than or equal to 50nM, greater than or equal to 100nM, greater than or equal to 500nM, greater than or equal to 1000nM, greater than or equal to 2000nM, greater than or equal to 3000nM, greater than or equal to 4000nM or greater than or equal to 5000 nM.

In some embodiments, an antigen binding molecule comprising an Fc region as described herein, which binds an activating Fc γ receptor (e.g., hfcyriia) selectively to an inhibitory Fc γ receptor (e.g., hfcyriib) is a peptide comprising SEQ ID NO: 174-175 the Fc region consisting of CH2-CH3 of the amino acid sequence shown in seq id No. has a binding selectivity greater than 1-fold, such as greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15-fold, or greater than 20-fold, for an equivalent antigen-binding molecule.

In some embodiments, an antigen binding molecule comprising an Fc region as described herein exhibits ADCC as a polypeptide comprising SEQ ID NO: 174-175 the Fc region consisting of CH2-CH3 of the amino acid sequence shown therein has greater than 1-fold, e.g., greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15-fold, or greater than 20-fold, ADCC for the equivalent antigen binding molecule.

In some embodiments, an antigen binding molecule comprising an Fc region as described herein, EC50(ng/ml) as determined in the assay for ADCC activity is a peptide comprising SEQ ID NO: 174, 175, CH2-CH3 of the amino acid sequence shown in seq id No. is less than 1-fold, e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2-fold, or less than 0.1-fold, of the equivalent antigen binding molecule determined EC50(ng/ml) of the Fc region.

In some embodiments, an antigen binding molecule comprising an Fc region as described herein has an EC50(ng/ml) of 500ng/ml or less, preferably one of 400ng/ml or less, 300ng/ml or less, 200ng/ml or less, 100ng/ml or less, 90ng/ml or less, 80ng/ml or less, 70ng/ml or less, 60ng/ml or less, 50ng/ml or less, 40ng/ml or less, 30ng/ml or less, 20ng/ml or less, or 10ng/ml in an assay for ADCC activity.

In some embodiments, the melting temperature, extension temperature, or decomposition temperature of an antigen binding molecule comprising an Fc region described herein can be a peptide comprising SEQ ID NO: 174-175 the equivalent antigen-binding molecule of the Fc region consisting of CH2-CH3 of the amino acid sequence shown in SEQ ID NO is 0.75 times or more and 1.25 times or less, e.g., 0.8 times or more and 1.2 times or less, 0.85 times or more and 1.15 times or less, 0.9 times or more and 1.1 times or less, 0.91 times or more and 1.09 times or less, 0.92 times or more and 1.08 times or less, 0.93 times or more and 1.07 times or less, 0.94 times or more and 1.06 times or more, 0.95 times or more and 1.05 times or more, 0.96 times or more and 1.04 times or more, 0.97 times or more and 1.03 times or more, 0.98 times or more and 1.02 times or more and 1.99 times or more and 1.01 times or less.

In some embodiments, the antigen binding molecules of the invention are capable of increasing killing of HER 3-expressing cells. Killing of HER3 expressing cells can be increased by the effector function of the antigen binding molecule. In embodiments where the antigen binding molecule comprises an Fc region, the antigen binding molecule may increase killing of HER3 expressing cells by one or more of Complement Dependent Cytotoxicity (CDC), antibody dependent cell mediated cytotoxicity (ADCC), and Antibody Dependent Cellular Phagocytosis (ADCP).

Antigen binding molecules capable of increasing killing of HER3 expressing cells can be identified by observing an increased level of killing on HER3 expressing cells in the presence of the antigen binding molecule or after incubation with HER3 expressing cells compared to the level of cell killing detected in the absence of the antigen binding molecule (or in the presence of an appropriate control antigen binding molecule) in an appropriate assay. Assays for CDC, ADCC and ADCP are well known to the skilled person. The level of killing of HER3 expressing cells can also be determined by measuring the number/ratio of viable and/or non-viable HER3 expressing cells after exposure to different treatment conditions.

In some embodiments, the antigen-binding molecules of the present invention are capable of increasing the killing of cells expressing HER3 (e.g., cancer cells expressing HER 3) to greater than 1-fold, e.g., greater than or equal to 1.01-fold, greater than or equal to 1.02-fold, greater than or equal to 1.03-fold, greater than or equal to 1.04-fold, greater than or equal to 1.05-fold, greater than or equal to 1.1-fold, greater than or equal to 1.2-fold, greater than or equal to 1.3-fold, greater than or equal to 1.4-fold, greater than or equal to 1.5-fold, greater than or equal to 1.6-fold, greater than or equal to 1.7-fold, greater than or equal to 1.8-fold, greater than or equal to 1.9-fold, greater than or equal to 2-fold, greater than or equal to 3-fold, greater than or equal to 4-fold, greater than or equal to 5-fold, greater than or equal to 6-fold, greater than or equal to 7-fold, greater than or equal to 8-fold, greater than or equal to 9-fold or greater than or 10-fold.

In some embodiments, an antigen-binding molecule of the invention is capable of reducing the number of cells expressing HER3 (e.g., cancer cells expressing HER 3) to at least less than 1 fold, e.g., less than or equal to 0.99 fold, less than or equal to 0.95 fold, less than or equal to 0.9 fold, less than or equal to 0.85 fold, less than or equal to 0.8 fold, less than or equal to 0.75 fold, less than or equal to 0.7 fold, less than or equal to 0.65 fold, less than or equal to 0.6 fold, less than or equal to 0.55 fold, less than or equal to 0.5 fold, less than or equal to 0.45 fold, less than or equal to 0.4 fold, less than or equal to 0.35 fold, less than or equal to 0.3 fold, less than or equal to 0.25 fold, less than or equal to 0.2 fold, less than or equal to 0.15 fold, less than or equal to 0.01 fold, less than or equal to 0.05 fold of the number of cells expressing HER3 (e.expressing cancer cells expressing HER3 expressing cancer cells) detected upon incubation in the absence of the antigen-binding molecule in a comparable experiment.

In some embodiments, the antigen binding molecules of the invention inhibit the development and/or progression of cancer in vivo.

In some embodiments, the antigen binding molecule causes increased killing of cancer cells, e.g., by effector immune cells. In some embodiments, the antigen binding molecule results in a reduction in the number of cancer cells in vivo, e.g., as compared to an appropriate control condition. In some embodiments, the antigen binding molecule inhibits tumor growth, for example, as determined by measuring tumor size/volume over time.

The antigen binding molecules of the invention can be assayed for their ability to inhibit cancer development and/or progression in an appropriate in vivo model (e.g., a cell line-derived xenograft model). Cell line-derived xenograft models can be derived from cancer cells that express HER 3. In some embodiments, the model is an N87 cell-derived model, an SNU16 cell-derived model, a FaDu cell-derived model, an OvCAR8 cell-derived model, an HCC95 cell-derived model, an a549 cell-derived model, an ACHN cell-derived model, or an HT29 cell-derived model.

The cancer may be a HER 3-associated cancer as described herein (i.e. a cancer in which HER3 gene/protein expression is a risk factor for the occurrence, development, progression or severity of symptoms and/or metastasis of the cancer, and/or is positively correlated therewith). The cancer may comprise cells expressing HER 3. In some embodiments, the cancer comprises HER3+ tumor.

In some embodiments, administration of an antigen binding molecule of the invention may result in one or more of the following: inhibiting the development/progression of cancer, delaying/preventing the onset of cancer, reducing/delaying/preventing the growth of a tumor, reducing/delaying/preventing metastasis, reducing the severity of cancer symptoms, reducing the number of tumor cells, reducing tumor size/volume, and/or increasing survival (e.g., progression-free survival), e.g., as determined in a transplant tumor model derived from a cancer cell line suitably expressing HER 3.

In some embodiments, in a transplanted tumor model derived from a cancer cell line that expresses HER3, the antigen binding molecules of the invention are capable of inhibiting tumor growth by less than 1 fold, e.g., by less than or equal to 0.99 times, by less than or equal to 0.95 times, by less than or equal to 0.9 times, by less than or equal to 0.85 times, by less than or equal to 0.8 times, by less than or equal to 0.75 times, by less than or equal to 0.7 times, by less than or equal to 0.65 times, by less than or equal to 0.6 times, by less than or equal to 0.55 times, by less than or equal to 0.5 times, by less than or equal to 0.45 times, by less than or equal to 0.4 times, by less than or equal to 0.35 times, by less than or equal to 0.3 times, by less than or equal to 0.25 times, by less than or equal to 0.2 times, by less than or equal to 0.15 times, by less than or equal to 0.1 times, by less than or equal to 0.05 times, in a case where no antigen binding molecule therapy is used.

Chimeric Antigen Receptors (CARs)

The invention also provides Chimeric Antigen Receptors (CARs) comprising the antigen binding molecules or polypeptides of the invention.

CARs are recombinant receptors that provide antigen binding and T cell activation functions. The structure and engineering of CARs is reviewed, for example, in Dotti et al, Immunol Rev (2014)257(1), the entire contents of which are incorporated herein by reference. The CAR comprises an antigen binding region linked to a cell membrane anchoring region and a signaling region. The optional hinge region may provide a separation between the antigen binding region and the cell membrane anchoring region and may serve as a flexible linker.

The CAR of the invention comprises an antigen binding region comprising or consisting of an antigen binding molecule of the invention, or comprising or consisting of a polypeptide of the invention.

The cell membrane-anchoring region is located between the antigen-binding region and the signaling region of the CAR for anchoring the CAR to the cell membrane of a cell expressing the CAR, wherein the antigen-binding region is located in the extracellular space and the signaling region is intracellular. In some embodiments, the CAR comprises a cell membrane anchoring region comprising or consisting of an amino acid sequence that comprises or consists of a transmembrane region amino acid sequence of one of CD 3-zeta, CD4, CD8, or CD 28. As used herein, a region "derived from" a reference amino acid sequence comprises an amino acid sequence that has at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the compared sequence.

The signaling region of the CAR can activate the T cell. The CAR signaling region can comprise the amino acid sequence of the intracellular domain of CD 3-zeta that provides an immunoreceptor tyrosine-based activation motif (ITAM) for phosphorylation and activation of CAR-expressing T cells. Signal regions comprising sequences of other ITAM-containing proteins, such as Fc γ RI, have also been used for CAR (Haynes et al, 2001J Immunol 166(1): 182-. The signaling region of the CAR can further comprise a co-stimulatory sequence derived from the signaling region of the co-stimulatory molecule to promote activation of the CAR-expressing T cell upon binding to the target protein. Suitable co-stimulatory molecules include CD28, OX40, 4-1BB, ICOS and CD 27. In certain instances, the CARs are engineered to co-stimulate different intracellular signaling pathways. For example, the signal transduction associated with co-stimulation of CD28 preferentially activates the phosphatidylinositol 3-kinase (P13K) pathway, whereas 4-1 BB-mediated signal transduction is via TNF Receptor Associated Factor (TRAF) adaptor proteins. Thus, the signaling region of a CAR sometimes includes co-stimulatory sequences derived from the signaling regions of more than one co-stimulatory molecule. In some embodiments, the CAR of the invention comprises one or more co-stimulatory sequences comprising or consisting of an amino acid sequence comprising, consisting of or derived from an amino acid sequence of an intracellular domain of one or more of: CD28, OX40, 4-1BB, ICOS and CD 27.

An optional hinge region may separate the antigen binding domain and the transmembrane domain, and may serve as a flexible linker. The hinge region may be derived from IgG 1. In some embodiments, the CAR of the invention comprises or consists of a hinge region comprising or consisting of an amino acid sequence that comprises or consists of an amino acid sequence of the hinge region of IgG 1.

Also provided are cells comprising the CARs of the invention. The CARs of the invention can be used to generate immune cells that express the CARs, such as CAR-T or CAR-NK cells. CAR can be engineered into immune cells during in vitro culture.

The antigen binding region of a CAR of the invention may be provided in any suitable form, e.g., scFv, scFab, etc.

Nucleic acids and vectors

The invention provides one or more nucleic acids encoding an antigen binding molecule, polypeptide, or CAR of the invention.

In some embodiments, the nucleic acid is, for example, purified or isolated from other nucleic acids or natural biological materials. In some embodiments, the one or more nucleic acids comprise or consist of DNA and/or RNA.

The invention also provides one or more vectors comprising one or more nucleic acids of the invention.

The nucleotide sequence may be comprised in a vector, such as an expression vector. As used herein, a "vector" is a nucleic acid molecule that serves as a vehicle for transferring exogenous nucleic acid into a cell. The vector may be a vector for expressing a nucleic acid in a cell. Such vectors may include a promoter sequence operably linked to a nucleotide sequence encoding the sequence to be expressed. The vector may also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer and stop codon known in the art may be used to express the peptide or polypeptide from the vectors of the invention.

The term "operably linked" may include situations in which a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and/or enhancer) are covalently linked in a manner that places expression of the nucleic acid sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if it is capable of affecting transcription of the nucleic acid sequence. The resulting transcript may then be translated into the desired peptide/polypeptide.

Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gamma retroviral vectors such as Murine Leukemia Virus (MLV) -derived vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and herpes viral vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes).

In some embodiments, the vector may be a eukaryotic vector, e.g., the vector comprises elements necessary for expression of the protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, e.g., comprising a Cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

The constituent polypeptides of the antigen binding molecules of the invention may be encoded by a variety of different nucleic acids, or by a variety of different vectors.

Cells comprising/expressing antigen binding molecules and polypeptides

The invention also provides a cell comprising or expressing an antigen binding molecule, polypeptide or CAR according to the invention. Also provided are cells comprising or expressing a nucleic acid, plurality of nucleic acids, vector or plurality of vectors of the invention.

The cell may be a eukaryotic cell, such as a mammalian cell. The mammal can be a primate (rhesus monkey, cynomolgus monkey, non-human primate or human) or a non-human mammal (e.g., a rabbit, guinea pig, rat, mouse or other rodent (including any animal of the order rodentia), cat, dog, pig, sheep, goat, cow (including a cow, e.g., cow, or any animal of the order bovinae), horse (including any animal of the order equines), donkey and non-human primate.

The invention also provides a method of producing a cell comprising one or more nucleic acids or vectors of the invention, comprising introducing a nucleic acid, nucleic acids, vector or vectors of the invention into a cell. In some embodiments, introducing one or more isolated nucleic acids or vectors of the invention into a cell comprises transformation, transfection, electroporation, or transduction (e.g., retroviral transduction).

The invention also provides a method of producing a cell expressing/comprising an antigen binding molecule, polypeptide or CAR according to the invention, comprising introducing into the cell a nucleic acid, nucleic acids, vector or vectors according to the invention. In some embodiments, the method further comprises culturing the cell under conditions suitable for expression of the nucleic acid or vector by the cell. In some embodiments, the method is performed in vitro.

The invention also provides a cell obtained or obtainable by the method of the invention.

Production of antigen binding molecules and polypeptides

The antigen binding molecules and polypeptides of the invention may be prepared according to methods for polypeptide production known to those skilled in the art.

The polypeptides may be prepared by chemical synthesis, e.g., liquid or solid phase synthesis. For example, peptides/polypeptides can be synthesized using methods such as those described in Chandrudu et al, Molecules (2013), 18:4373-4388, which is incorporated herein by reference in its entirety.

Alternatively, the antigen binding molecules and polypeptides may be produced by recombinant expression. Molecular biology techniques suitable for recombinant production of polypeptides are well known in the art, for example Green and Sambrook, molecular cloning: a Laboratory Manual (4 th edition), Cold spring harbor Press, 2012, and Nat Methods, (2008)5(2): 135-. Methods for recombinant production of antigen binding molecules are also described in Frenzel et al, Front Immunol (2013); 4:217 and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016)100: 3451-3461, both of which are incorporated herein by reference in their entirety.

In certain instances, the antigen binding molecules of the invention are comprised of more than one polypeptide chain. In such cases, the production of the antigen binding molecule may include transcription and translation of more than one polypeptide, and subsequent binding of the polypeptide chains to form the antigen binding molecule.

For recombinant production according to the invention, any cell suitable for expressing a polypeptide may be used. The cell may be a prokaryote or a eukaryote. In some embodiments, the cell is a prokaryotic cell, such as an archaebacterium or bacterial cell. In some embodiments, the bacterium can be a gram-negative bacterium, such as a bacterium of the enterobacteriaceae family, e.g., e. In some embodiments, the cell is a eukaryotic cell, such as a yeast cell, a plant cell, an insect cell, or a mammalian cell, such as a CHO, HEK (e.g., HEK293), HeLa, or COS cell. In some embodiments, the cell is a CHO cell that transiently or stably expresses the polypeptide.

In some cases, the cell is not a prokaryotic cell, as some prokaryotic cells do not allow the same folding or post-translational modifications as eukaryotic cells. Furthermore, there may be very high expression levels in eukaryotes, and proteins can be more easily purified from eukaryotes using appropriate tags. Specific plasmids which enhance secretion of the protein into the culture medium may also be used.

In some embodiments, the polypeptide can be synthesized by cell-free protein synthesis (CFPS), for example, using Zemella et al, Chembiochem (2015)16 (17): 2420 the system preparation described in 2431, which is incorporated herein by reference in its entirety.

Production may involve culturing or fermenting a eukaryotic cell modified to express the polypeptide of interest. The cultivation or fermentation can be carried out in a bioreactor equipped with appropriate nutrients, air/oxygen and/or growth factors. Secreted proteins can be collected by isolating the culture medium/fermentation broth from the cells, extracting the protein content, isolating each protein to isolate the secreted polypeptide. Culture, fermentation and isolation techniques are well known to those skilled in the art and are described, for example, in Green and Sambrook, molecular cloning: a laboratory Manual (4 th edition; incorporated herein by reference).

The bioreactor includes one or more vessels in which cells can be cultured. The culture in the bioreactor may be carried out continuously, wherein reactants are continuously flowed into the reactor and cultured cells are continuously flowed out of the reactor. Alternatively, the cultivation may be carried out batchwise. The bioreactor monitors and controls environmental conditions such as pH, oxygen, flow rates into and out of the vessel, and agitation within the vessel to provide optimal conditions for the cultured cells.

After culturing the cells expressing the antigen binding molecule/polypeptide, the polypeptide of interest can be isolated. Any suitable method known in the art for isolating proteins from cells may be employed. In order to isolate the polypeptide, it may be necessary to separate the cells from the nutrient medium. If the one or more polypeptides are secreted by the cells, the cells can be separated from the medium containing the secreted polypeptide or polypeptides of interest by centrifugation. If one or more polypeptides of interest are aggregated within the cell, protein isolation may include centrifugation to separate the cells from the cell culture medium, treatment of the cell pellet with lysis buffer, and cell disruption, such as by sonication, rapid freeze-thawing, or osmotic lysis.

It may then be desirable to isolate the one or more polypeptides of interest from the supernatant or culture medium, which may contain other proteinaceous and non-proteinaceous components. A common method for separating the protein component from the supernatant or the culture medium is precipitation. Different concentrations of precipitating agents, such as ammonium sulfate, precipitate proteins of different solubilities. For example, low concentrations of precipitating agents extract water soluble proteins. Thus, by adding different concentrations of increasing precipitating agent, proteins with different solubilities can be distinguished. Dialysis can then be used to remove ammonium sulfate from the isolated protein.

Other methods of distinguishing between different proteins are known in the art, such as ion exchange chromatography and size chromatography. These may be used as an alternative to precipitation, or may be carried out after precipitation.

Once the one or more polypeptides of interest are isolated from the culture, it may be desirable or necessary to concentrate the one or more polypeptides. Many methods of concentrating proteins are known in the art, such as ultrafiltration or lyophilization.

Composition comprising a fatty acid ester and a fatty acid ester

The invention also provides compositions comprising the antigen binding molecules, polypeptides, CARs, nucleic acids, expression vectors, and cells described herein.

The antigen binding molecules, polypeptides, CARs, nucleic acids, expression vectors, and cells described herein can be formulated into pharmaceutical compositions or medicaments for clinical use and can include pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The compositions may be formulated for topical, parenteral, systemic, intracavity, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration, including injection or infusion.

Suitable formulations may comprise the antigen binding molecule in a sterile or isotonic medium. The medicaments and pharmaceutical compositions may be formulated in fluid form, including gels. The fluid formulation may be formulated for administration to a selected region of the human or animal body by injection or infusion (e.g. via a catheter).

In some embodiments, the composition is formulated for injection or infusion, e.g., into a blood vessel or tumor.

According to the invention described herein, there is also provided a process for the production of a pharmaceutically useful composition, such production process may comprise one or more steps selected from: producing an antigen binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell as described herein; isolating an antigen binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell described herein; and/or admixing an antigen binding molecule, polypeptide, CAR, nucleic acid (or nucleic acids), expression vector (or expression vectors), or cell as described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

For example, another aspect of the invention described herein relates to a method of formulating or producing a medicament or pharmaceutical composition for treating a disease/disorder (e.g., cancer) comprising formulating the pharmaceutical composition by mixing an antigen binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

Therapeutic and prophylactic applications

The antigen binding molecules, polypeptides, CARs, nucleic acids, expression vectors, cells, and compositions described herein can be used in therapeutic and prophylactic methods.

The invention provides an antigen binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition described herein for use in a medical treatment or prevention method. Also provided is the use of an antigen binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition described herein in the manufacture of a medicament for the treatment or prevention of a disease or disorder. Also provided is a method of treating or preventing a disease or disorder comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition described herein.

The methods are effective to reduce the development or progression of the disease/disorder, alleviate symptoms of the disease/disorder, or alleviate the pathological characteristics of the disease/disorder. The methods may be effective in preventing the progression of the disease/disorder, for example preventing the worsening of the disease/disorder or slowing its rate of progression. In some embodiments, the methods may result in an improvement in the disease/disorder, such as a reduction in the symptoms of the disease/disorder or a reduction in some other relevant characteristic of the severity/activity of the disease/disorder. In some embodiments, the methods can prevent the development of a disease/disorder in a later stage (e.g., chronic stage or metastasis).

It will be appreciated that the preparation of the invention may be used to treat/prevent any disease/disorder for which a therapeutic or prophylactic benefit would be derived from a reduction in the number and/or activity of cells expressing HER 3. For example, the disease/disorder may be a disease/disorder in which cells expressing HER3 are pathologically involved, such as a disease/disorder in which an increase in the number/proportion of cells expressing HER3 is directly correlated with the onset, occurrence or progression of the disease/disorder and/or the severity of one or more symptoms of the disease/disorder, or a disease/disorder in which an increase in the number/proportion of cells expressing HER3 is a risk factor for the onset, occurrence or progression of the disease/disorder.

In some embodiments, the disease/disorder to be treated/prevented according to the invention is a disease/disorder characterized by an increased number/ratio/activity of cells expressing HER3, e.g. compared to the number/ratio/activity of cells expressing HER3 in the absence of the disease/disorder.

In some embodiments, the disease/disorder to be treated/prevented is cancer.

A cancer can be any undesirable cell proliferation (or any disease that manifests itself in undesirable cell proliferation), neoplasm, or tumor. Cancer can be benign or malignant, and can be primary or secondary (metastatic). A neoplasm or tumor can be any abnormal growth or proliferation of a cell and can be located in any tissue. The cancer may be tissue/cells derived from, for example, adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone marrow, brain, breast, cecum, central nervous system (including or not including brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelium), gall bladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph nodes, lymphoblasts, maxilla, mediastinum, mesenterium, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid, tongue, tonsil, trachea, uterus, vulva, and/or leukocytes.

The tumor to be treated may be a neural or non-neural tumor. Tumors of the nervous system may originate from the central or peripheral nervous system, such as gliomas, medulloblastomas, meningiomas, neurofibromas, ependymomas, schwannomas, neurofibrosarcomas, astrocytomas and oligodendrogliomas. Non-nervous system cancers/tumors may originate from any other non-nervous tissue, including for example melanoma, mesothelioma, lymphoma, myeloma, leukemia, non-hodgkin's lymphoma (NHL), hodgkin's lymphoma, Chronic Myelogenous Leukemia (CML), Acute Myelogenous Leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), Chronic Lymphocytic Leukemia (CLL), liver cancer, epidermoid cancer, prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymus cancer, NSCLC, hematological cancer and sarcoma.

HER3 and its relationship to and role in cancer in Karachaliou et al, biopharmaceuticals (2017)31 (1): 63-73 and Zhang et al, Acta Biochimica et Biophysica Sinica (2016)48 (1): 39-48, both of which are incorporated herein by reference in their entirety.

In some embodiments, the cancer is selected from: comprising a HER 3-expressing cell, a solid tumor, breast cancer, breast tumor, ductal cancer, gastric tumor, gastric adenocarcinoma, colorectal cancer, colorectal tumor, colorectal adenocarcinoma, head and neck cancer, head and neck Squamous Cell Carcinoma (SCCHN), lung cancer, lung adenocarcinoma, squamous cell lung cancer, ovarian tumor, ovarian serous adenocarcinoma, kidney cancer, kidney cell tumor, renal clear cell carcinoma, kidney cell adenocarcinoma, renal papillary cell carcinoma, pancreatic cancer, pancreatic tumor, pancreatic ductal adenocarcinoma, cervical cancer, cervical squamous cell carcinoma, skin cancer, melanoma, esophageal cancer, esophageal adenocarcinoma, liver cancer, hepatocellular carcinoma, bile duct cancer, uterine cancer, endometrial cancer, thyroid tumor, pheochromocytoma, paraganglioma, bladder cancer, urinary bladder urothelial cancer, prostate cancer, sarcoma, and thymoma.

In some embodiments, the cancer treated according to the invention is selected from: HER 3-expressing cancer, gastric cancer (e.g. gastric tumor, gastric adenocarcinoma, gastrointestinal adenocarcinoma), head and neck cancer (e.g. head and neck squamous cell carcinoma), breast cancer, ovarian cancer (e.g. ovarian tumor), lung cancer (e.g. NSCLC, lung adenocarcinoma, squamous cell carcinoma), melanoma, prostate cancer, oral cancer (e.g. oropharyngeal cancer), renal cancer (e.g. renal cell carcinoma) or large bowel cancer (e.g. large bowel tumor), esophageal cancer, pancreatic cancer, solid cancer and/or liquid cancer.

The treatment/prevention may be for one or more of: delaying/preventing onset/progression of cancer symptoms, reducing severity of cancer symptoms, reducing survival/growth/invasion/metastasis of cancer cells, reducing number of cancer cells, and/or increasing survival rate of a subject.

In some embodiments, the cancer to be treated/prevented comprises cells expressing an EGFR family member (e.g., HER3, EGFR, HER2, or HER4), and/or cells expressing an EGFR family member ligand. In some embodiments, the cancer to be treated/prevented is a cancer that is positive for an EGFR family member. In some embodiments, the cancer overexpresses an EGFR family member and/or a ligand for an EGFR family member. Overexpression can be determined by measuring the expression level, which is higher than that of an equivalent non-cancerous cell/non-tumor tissue.

Expression may be determined by any suitable means. Expression may be gene expression or protein expression. Gene expression can be determined by, for example, quantitative real-time PCR (qRT-PCR) detection of mRNA encoding HER 3. Protein expression can be determined, for example, by antibody-based methods such as western blot, immunohistochemistry, immunocytochemistry, flow cytometry or ELISA.

In some embodiments, the cancer to be treated/prevented comprises a cell expressing HER 3. In some embodiments, the cancer to be treated/prevented is a HER3 positive cancer. In some embodiments, the cancer overexpresses HER 3. Overexpression of HER3 can be determined by detecting the expression level of HER3, which is greater than the expression level of equivalent non-cancer cells/non-tumor tissues.

In some embodiments, a patient is selected for treatment described herein based on detecting a HER3 expressing cancer or a HER3 overexpressing cancer, e.g., in a sample obtained from a subject.

In some embodiments, the cancer to be treated/prevented comprises cells expressing a ligand of HER3 (e.g., NRG1 and/or NRG 2). In some embodiments, the cancer to be treated/prevented comprises cells that express NRG1 and/or NRG2 at a level higher than the expression level of equivalent non-cancerous cells/non-tumor tissue.

The antigen binding molecules described herein that bind HER3 were demonstrated to bind HER3 with very high affinity when HER3 is bound by NRG (i.e. when HER3 is provided in an "open" conformation) and when HER3 is not bound by NRG (i.e. when HER3 is provided in a "closed" conformation).

Thus, the antigen binding molecules of the present invention are particularly useful for the treatment/prevention of cancers characterized by HER3 ligand expression/overexpression, such as cancers/tumors comprising cells expressing/overexpressing HER3 ligand.

In some embodiments, the cancer to be treated according to the invention comprises cells carrying a genetic variant (e.g., a mutation) that results in increased expression of HER3 ligand (gene and/or protein) relative to comparable cells carrying a reference allele that does not comprise the genetic variant (e.g., an unmutated or "wild-type" allele). The genetic variant may be or include an insertion, deletion, substitution or larger scale translocation/rearrangement of the nucleotide sequence relative to the reference allele.

A mutation that "causes" an increase in HER3 ligand expression may be known or predicted to cause an increase in gene/protein expression of HER3 ligand, or may be associated with an increase in gene/protein expression of HER3 ligand. Mutations that result in increased expression of HER3 ligand may be referred to as "activating" mutations.

Mutations that result in increased expression of HER3 ligand may result in expression of a gene or protein of HER3 ligand that is not expressed by, and/or is not encoded by, the genomic nucleic acid of the equivalent cell that does not carry the mutation. That is, the ligand of HER3 may be a neoantigen due to mutation, and thus "increased expression" may be due to no expression. For example, a cell comprising a CD47-NRG1 gene fusion exhibits increased expression of a CD47-NRG1 fusion polypeptide encoded by the gene fusion relative to a cell lacking a CD47-NRG1 gene fusion.

A mutation that results in increased expression of a HER3 ligand may result in increased expression of a gene or protein of HER3 ligand expressed by and/or encoded by the genomic nucleic acid of an equivalent cell that does not comprise the mutation. For example, the cell may comprise a mutation that results in an increase in the level of transcription of a nucleic acid encoding NRG1 relative to the level of transcription of an equivalent cell nucleic acid encoding NRG1 that does not comprise the mutation.

In some embodiments, a mutation that results in increased expression of HER3 ligand may result in increased gene expression of HER3 ligand relative to an equivalent cell that does not comprise the mutation. In some embodiments, a mutation that results in increased expression of HER3 ligand may result in increased protein expression of HER3 ligand relative to an equivalent cell that does not comprise the mutation.

In some embodiments, a mutation that results in increased expression of HER3 ligand may result in an increase in HER3 ligand levels on or at the cell surface comprising the mutation relative to an equivalent cell that does not comprise the mutation. In some embodiments, a mutation that results in increased expression of HER3 ligand may result in a cell comprising the mutation increasing the level of secretion of HER3 ligand relative to an equivalent cell that does not comprise the mutation.

A cell with increased expression of HER3 ligand (e.g., due to mutation) relative to the expression level of HER3 ligand of a reference cell may be described as HER3 ligand "overexpressing", or having "upregulated expression" of HER3 ligand. For example, a cancer comprising a cell in which the mutation results in increased HER3 ligand expression relative to an equivalent cell lacking the mutation may be described as a cancer comprising a cell in which HER3 ligand is overexpressed/upregulated. In some embodiments, a reference cell lacking a mutation can be a non-cancerous cell (e.g., of an equivalent cell type) or a cancerous cell (e.g., of an equivalent cancer type).

Herein, "HER 3 ligand" generally refers to a molecule capable of binding to HER3 through the ligand binding region of HER3 formed by domains I and III of HER 3. In some embodiments, a ligand of HER3 binds to HER3 by interacting with domain I and/or III of HER 3. Exemplary ligands for HER3 include neuregulin proteins such as NRG1 and NRG2, which bind to HER3 through the interaction between their EGF-like domains and the ligand-binding region of HER 3.

HER3 ligand is preferably able to bind and trigger signaling through HER3 receptor and/or a receptor complex comprising HER 3. It will be clear from the present disclosure that a receptor complex comprising HER3 may further comprise an interaction partner of HER3 as described herein, e.g. HER3, HER2, EGFR, HER4, HGFR, IGF1R and/or cMet).

In some embodiments, the ligand of HER3 is capable of binding to a cell expressing a HER3 receptor/receptor complex rather than a cell with increased expression of HER3 ligand. For example, in some embodiments, a ligand of HER3 is capable of binding to a cancer cell expressing HER 3.

In some embodiments, the ligand of HER3 is capable of binding to the HER3 receptor/receptor complex expressed by cells with increased expression of HER3 ligand.

In some embodiments, the cancer to be treated comprises (i) a cell expressing HER3, and (ii) a cell expressing HER3 ligand (e.g. increased expression of HER3 ligand in the cell, e.g. increased expression of HER3 ligand due to mutation).

In some embodiments, the cancer to be treated comprises (i) a cell that expresses HER3 and (ii) a cell that also expresses HER3 ligand (e.g., increased expression of HER3 ligand in the cell, e.g., increased expression of HER3 ligand due to a mutation).

In some embodiments, the HER3 ligand comprises, or consists of, the amino acid sequence of the HER3 binding region of HER3 ligand, or the amino acid sequence of the HER3 binding region derived from HER3 ligand. The amino acid sequence of the HER3 binding region derived from a HER3 ligand may comprise at least 60% (e.g. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to its derived amino acid sequence.

In some embodiments, the ligand of HER3 comprises an EGF-like domain capable of binding to HER3, or a HER3 binding fragment thereof. In some embodiments, HER3 binding EGF-like domain/fragment is or is derived from an EGF family member (e.g., heparin binding EGF-like growth factor (HB-EGF), transforming growth factor-alpha (TGF-alpha), dual regulatory protein (AR), epidermal regulator (EPR), epidermal growth factor (epigen), cytokine (BTC), NRG1, NRG2, NRG3, or NRG 4).

EGF family members comprise one or more repeats of a conserved amino acid sequence shown in SEQ ID NO:240, containing 6 cysteine residues, forming 3 intramolecular disulfide bonds, providing 3 structural loops that require high affinity binding to their cognate receptors (see Harris et al, Experimental cell research (2003)284(1): 2-13). In some embodiments, the ligand of HER3 comprises one or more copies of an amino acid sequence conforming to the consensus sequence set forth in SEQ ID NO: 240.

Exemplary ligands for HER3 include Neuregulin (NRG). Neuregulin proteins include NRG1, NRG2, NRG3 and NRG 4. The amino acid sequence of human NRG1 (alpha isoform) is shown in SEQ ID NO: 232. The α isoform and several other isoforms of human NRG1, including the α 1a isoform (see UnitProt: Q02297-2), the α 2b isoform (see UnitProt: Q02297-3) and the α 3 isoform (see UnitProt: Q02297-4), contain the EGF-like domain shown in SEQ ID NO:233, through which it binds to HER 3. The amino acid sequence of human NRG2 (isoform 1) is shown in SEQ ID NO: 234. Isoform 1 and several other isoforms of human NRG2, including isoform 3 (see Uniprot: O14511-3), isoform 5 (see Uniprot: O14511-5), isoform 6 (see Uniprot: O14511-6), isoform DON-1B (see Uniprot: O14511-7) and isoform DON-1R (see Uniprot: O14511-8), contain an EGF-like domain as shown in SEQ ID NO:235, through which it binds to HER 3. The amino acid sequence of human NRG3 is shown in SEQ ID NO 236, and the EGF-like domain of human NRG3 is shown in SEQ ID NO 237. The amino acid sequence of human NRG4 is shown in SEQ ID NO. 238, and the EGF-like domain of human NRG3 is shown in SEQ ID NO. 239. In some embodiments, the NRG is selected from NRG1, NRG2, NRG3, and NRG 4. In some embodiments, the NRG is selected from NRG1 and NRG 2.

In some embodiments, the EGF-like domain/fragment comprises or consists of an amino acid sequence having at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to the EGF-like domain of NRG (NRG1, NRG2, NRG3, or NRG 4). In some embodiments, the EGF-like domain/fragment comprises or consists of an amino acid sequence having at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to one of SEQ ID NOs 233, 235, 237, or 239.

In some embodiments, the ligand of HER3 is NRG (e.g., NRG1, NRG2, NRG3, or NRG 4; e.g., NRG1 or NRG2), or an amino acid sequence comprising an amino acid sequence derived from NRG (i.e., an amino acid sequence comprising at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to an amino acid sequence of NRG.

In some embodiments, a ligand of HER3 comprises or consists of an amino acid sequence that has at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to a HER3 binding region of HER3 ligand (e.g., NRG, such as NRG1, NRG2, NRG3 or NRG 4; such as NRG1 or NRG 2). In some embodiments, the ligand of HER3 comprises or consists of an amino acid sequence that has at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to an EGF-like domain of NRG (e.g., NRG1, NRG2, NRG3 or NRG 4; e.g., NRG1 or NRG 2).

In some embodiments, the ligand of HER3 is not an EGFR family protein (e.g., HER3, HER2, EGFR, HER4, HGFR, IGF1R, cMet).

In some embodiments, the mutation that results in increased expression of HER3 ligand is an NRG gene fusion. In some embodiments, the ligand of HER3 is the product of (i.e., the polypeptide encoded by) a NRG gene fusion. In some embodiments, the cancer comprises a cell having NRG gene fusion.

As used herein, "NRG gene fusion" refers to a genetic variant that encodes a polypeptide comprising (i) the amino acid sequence of an NRG protein (e.g., NRG1, NRG2, NRG3 or NRG 4; e.g., NRG1 or NRG2) and (ii) the amino acid sequence of a protein other than an NRG protein.

It will be appreciated that NRG gene fusions preferably encode HER3 ligands as described herein. In some embodiments, the NRG gene fusion encodes a polypeptide comprising a HER3 binding region of an NRG protein. In some embodiments, the NRG gene fusion encodes a polypeptide comprising an EGF-like domain of the NRG protein or an amino acid sequence capable of binding to HER3 and having at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the EGF-like domain of the NRG protein.

In some embodiments, the NRG gene fusion encodes a fusion polypeptide comprising a transmembrane domain. In some embodiments, the NRG gene fusion encodes a fusion polypeptide comprising a transmembrane domain of a protein other than an NRG protein.

In some embodiments, the NRG gene fusion is an NRG1 gene fusion. In some embodiments, the NRG1 gene fusion encodes a polypeptide comprising an EGF-like domain of NRG1 or an amino acid sequence capable of binding to HER3 and having at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the EGF-like domain of NRG 1.

NRG1 gene fusions are described, for example, in WO 2018/182422 a1, WO 2019/051155 a1, Dhanasekaran et al, Nat Commun. (2014)5:5893, Drilon et al, Cancer Discov. (2018)8 (6): 686-: 1354 and Jonna et al, Clin Cancer Res. (2019)25(16):4966 and 4972, all of which are incorporated herein by reference in their entirety. The diversity of NRG1 gene fusions may be due to the fact that NRG1 is located on chromosome 8 and is thus particularly susceptible to genomic translocation events

Et al, Genes Chromosomes Cancer, (2003)37(4): 333-45).

In some embodiments, the NRG gene fusion is selected from the group consisting of CLU-NRG, CD-NRG, DOC-NRG, SLC 3A-NRG, RBPMS-NRG, WRN-NRG, SDC-NRG, RAB2 IL-NRG, VAMP-NRG, KIF 13-NRG, THAP-NRG, SMAD-NRG, MDK-NRG, TNC-NRG, DIP 2-NRG, MRPL-NRG, PARP-NRG, ROCK-NRG, DPYSL-NRG, ATP 1B-NRG, CDH-NRG, APP-NRG, AKAP-NRG, THBS-NRG, FOXA-NRG, PDE 7-NRG, RAB3 IL-NRG, CDK-NRG, BMPRIB-NRG, TNFRSF 10-NRG, and MCPH-NRG. In some embodiments, the NRG1 gene fusion is CLU-NRG 1.

CD74-NRG1 gene fusions are described in Fernandez-Cuesta et al, Cancer Discov. (2014)4:415-22 and Nakaoku et al, Clin Cancer Res (2014)20: 3087-93. DOC4-NRG1 gene fusions are described, for example, in Liu et al, Oncogene (1999)18 (50): 7110-4 and Wang et al, Oncogene, (1999)18(41): 5718-21. SLC3A2-NRG1 gene fusions are described, for example, in Nakaoku et al, Clin Cancer Res (2014)20:3087-93, Shin et al, Oncotarget (2016)7:69450-65 and Shin et al, Mol Cancer Ther (2018)17(9): 2024-. RBPMS-NRG1, WRN-NRG1, RAB2IL1-NRG1 and SDC4-NRG1 gene fusions are described, for example, in Dhanasekaran et al, Nat Commun, (2014)5: 5893. VAMP2-NRG1 gene fusions are described, for example, in Jung et al, J Thorac Oncol. (2015)10 (7): 1107-11 and Shim et al, J Thorac Oncol (2015)10(8): 1156-62. KIF13B-NRG1 gene fusions are described, for example, in Xia et al, Int J Surg Pathol (2017)25(3): 238-240. SMAD4-NRG1, AKAP13-NRG1, THBS1-NRG1, FOXA1-NRG1, PDE7A-NRG1, RAB3IL1-NRG1 and THAP7-NRG1 gene fusions are described, for example, in Drilon et al, Cancer DiscoV (2018)8(6) 686-. MDK-NRG1, TNC-NRG1, DIP2B-NRG1, MRPL13-NRG1, PARP8-NRG1, ROCK1-NRG1 and DPYSL2-NRG1 gene fusions are described, for example, in Jonna et al, Clin Cancer Res. (2019)25(16):4966 and 4972. ATP1B1-NRG1 gene fusions are described, for example, in Drilon et al, Cancer Discov. (2018)8(6): 686-. CLU-NRG1 gene fusions are described, for example, in Drilon et al, Cancer Discov. (2018)8(6): 686. abone 695 and Nagasaka et al, Journal of clinical Oncology (2019)14(8): 1354. abone 1359.

In some embodiments, the NRG gene fusion is an NRG2 gene fusion. In some embodiments, the NRG2 gene fusion encodes a polypeptide comprising an EGF-like domain of NRG2 or an amino acid sequence capable of binding to HER3 and having at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the EGF-like domain of NRG 2.

NRG2 gene fusions include SLC12A2-NRG2, described, for example, in WO 2015/093557A 1, and ZNF208-NRG2, described, for example, in Dupain et al, Mol Ther, (2019)27(1) 200-.

A cancer comprising a cell with a mutation that results in increased expression of a HER3 ligand (e.g., a cell comprising a fusion with a NRG gene, such as a NRG1 gene fusion or a NRG2 gene fusion) can be any cancer described herein. In some embodiments, the cancer may be a tissue/cell derived from lung, breast, head, neck, kidney, ovary, pancreas, prostate, uterus, gall bladder, colon, rectum, bladder, soft tissue, or nasopharynx.

In some embodiments, a cancer comprising a cell with a mutation that results in increased expression of a HER3 ligand (e.g., a cell comprising a fusion with a NRG gene, such as a NRG1 gene fusion or a NRG2 gene fusion) is selected from the group consisting of: lung cancer, non-small cell lung cancer, lung adenocarcinoma, aggressive lung mucus adenocarcinoma, lung squamous cell carcinoma, breast cancer, breast tumor, breast invasive carcinoma, head and neck cancer, head and neck squamous cell carcinoma, kidney cancer, renal clear cell carcinoma, ovarian cancer, ovarian serous cystadenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, prostate cancer, prostate adenocarcinoma, endometrial carcinoma, uterine carcinosarcoma, gallbladder cancer, bile duct cancer, colorectal cancer, bladder cancer, urothelial bladder cancer, sarcoma, soft tissue sarcoma, neuroendocrine tumor, and neuroendocrine tumor of the nasopharynx.

In a particular embodiment, the cancer to be treated according to the invention is lung cancer (e.g. non-small cell lung cancer, lung adenocarcinoma, aggressive lung mucinous adenocarcinoma or lung squamous cell carcinoma) comprising cells with a fusion of the NRG1 gene.

It is understood that in embodiments herein, a cancer comprising cells having a particular characteristic may be or include a tumor comprising cells having such characteristics.

As is common in the art, a cancer/tumor comprising cells having a particular characteristic may be referred to herein simply as a cancer/tumor having such characteristics. By way of illustration, a cancer/tumor comprising a cell with NRG1 gene fusion may be referred to simply as a "cancer/tumor comprising NRG1 gene fusion", or a "cancer/tumor of NRG1 gene fusion".

Administration of the preparations of the invention is preferably administered in a "therapeutically effective" or "prophylactically effective" amount, sufficient to show a therapeutic or prophylactic benefit to the subject. The amount actually administered, as well as the rate and time course of administration, will depend on the nature and severity of the disease/disorder and the particular substance being administered. The decision on the prescription of treatment, e.g. dosage, etc., is at the responsibility of general practitioners and other medical doctors, and generally takes into account the disease/disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of such techniques and protocols can be found in Remington's Pharmaceutical Sciences, 20 th edition, 2000, published by RipIkett, Williams and Wilkins publishers.

Depending on the disease to be treated, administration may be separate or in combination with other therapeutic methods, either simultaneously or sequentially. The antigen binding molecule or composition described herein and the therapeutic agent can be administered simultaneously or sequentially.

In some embodiments, the methods comprise additional therapeutic or prophylactic intervention, e.g., for treating/preventing cancer. In some embodiments, the therapeutic or prophylactic intervention is selected from chemotherapy, immunotherapy, radiation therapy, surgery, vaccination, and/or hormonal therapy. In some embodiments, the therapeutic or prophylactic intervention comprises leukapheresis. In some embodiments, the therapeutic or prophylactic intervention comprises stem cell transplantation.

In some embodiments, the antigen binding molecule is administered in combination with an agent capable of inhibiting EGFR family member-mediated signaling.

Accordingly, the invention provides a composition comprising an article of manufacture of the invention (e.g., an antigen binding molecule according to the invention) and another agent capable of inhibiting signaling mediated by an EGFR family member (e.g., EGFR, HER2, HER3, or HER 4). Also provided is the use of such compositions in the methods of medical treatment and prevention of diseases/disorders described herein.

Also provided are methods for treating/preventing a disease/disorder described herein, comprising administering an article of manufacture of the invention (e.g., an antigen binding molecule of the invention) and another agent capable of inhibiting signaling mediated by an EGFR family member.

Agents capable of inhibiting EGFR family members mediated signal transduction are known in the art and include: such as small molecule inhibitors (e.g., tyrosine kinase inhibitors), monoclonal antibodies (and antigen binding fragments thereof), peptide/polypeptide inhibitors (e.g., decoy ligands/receptors or peptide aptamers), and nucleic acids (e.g., antisense nucleic acids, splice switching nucleic acids, or nucleic acid aptamers). Inhibitors of EGFR family member-mediated signal transduction include agents that act directly on EGFR family members, and their interaction partners, and/or agents that participate in downstream factors of signal transduction mediated by EGFR family members.

In some embodiments, the antagonist of signaling mediated by an EGFR family member inhibits signaling mediated by one or more of EGFR, HER2, HER4, and HER 3. Inhibitors of signaling mediated by EGFR family members are described in Yamaoka et al, int.j.mol.sci. (2018), 19, 3491, the entire contents of which are incorporated herein by reference. In some embodiments, the antagonist is a pan-ErbB inhibitor. In some embodiments, the antagonist is an inhibitor of EGFR-mediated signaling (e.g., cetuximab (cetuximab), panitumumab (panitumumab), gefitinib (gefitinib), erlotinib (erlotinib), lapatinib (lapatinib), afatinib (afatinib), brigatinib (brigatinib), etitinib (icotinib), ocitinib (osimertinib), zalutumumab (zalutumumab), vandetanib (vandetanib), tuzumab (necitumumab), nimotuzumab (nimotuzumab), dacomitinib (dacomitinib), deglutumumab (duliguzumab), or matuzumab (matuzumab)). In some embodiments, the antagonist is an inhibitor of HER 2-mediated signaling (e.g., trastuzumab (trastuzumab), pertuzumab (pertuzumab), lapatinib (lapatinib), neratinib (neratinib), afatinib (afatinib), dacomizumab (dacomitinib), MM-111, MCLA-128, or magituximab (margetuximab). in some embodiments, the antagonist is an inhibitor of HER 3-mediated signaling (e.g., selituzumab (seribab), lumecritumumab (lumretuzumab), eggemumab (elgimatinib), KTN3379, AV-203, GSK2849330, REGN1400, MP-RM-1, EV20, degugutezumab (duliguzumab), iruzumab-111, epritumumab (irist), mck 2849330, mck EZN, or an antagonist of HER 361402-EZN-mediated signaling (e.g., in 361402, EZN) Ibrutinib (ibrutinib), afatinib (afatinib), dacomitinib (dacomitinib) or neratinib).

In some embodiments, the antagonist of signaling mediated by an EGFR family member inhibits a downstream signaling effector of the EGFR family member. Downstream effectors of EGFR family member signaling include: such as PI3K, AKT, KRAS, BRAF, MEK/ERK, and mTOR. In some embodiments, the antagonist of signaling mediated by an EGFR family member is an inhibitor of the MAPK/ERK pathway. In some embodiments, the antagonist of signaling mediated by an EGFR family member is an inhibitor of the PI3K/ATK/mTOR pathway. In some embodiments, the antagonist is a PI3K inhibitor (e.g., picrolisib (pictiliib), bupirisib (buparlisib), idelalisib (idelalisib), coppanisib (copanlisib), or duvelisib). In some embodiments, the antagonist is an AKT inhibitor (e.g., MK-2206, AZD5363, estasertib, VQD-002, perifosine, or miltefosine). In some embodiments, the antagonist is a BRAF inhibitor (e.g., vemurafenib (vemurafenib), dabrafenib (dabrafenib), SB590885, XL281, RAF265, enrafenib (encorafenib), GDC-0879, PLX-4720, sorafenib (sorafenib), or LGX 818). In some embodiments, the antagonist is a MEK/ERK inhibitor (e.g., trametinib (trametinib), cobinetinib (cobimetinib), bimetinib (binimetinib), semetinib (selumetinib), PD-325901, CI-1040, PD035901, or TAK-733). In some embodiments, the antagonist is an mTOR inhibitor (e.g., rapamycin (rapamycin), deformolimus (deforolimus), temsirolimus (temsirolimus), everolimus (everolimus), ridaforolimus (ridaforolimus), or sappanisissib).

In some embodiments, the cancer to be treated according to one aspect of the invention (including monotherapy or combination therapy) is resistant to treatment with an antagonist of signaling mediated by an EGFR family member (e.g., EGFR, HER2, HER4, and/or HER3) (e.g., an antagonist as described in the preceding three paragraphs). In some embodiments, the cancer of the subject to be treated is resistant to treatment with a signaling antagonist mediated by an EGFR family member. In some embodiments, the cancer of the subject to be treated has developed resistance to treatment with a signaling antagonist mediated by an EGFR family member. In some embodiments, the cancer of the subject to be treated has previously responded to treatment with a signal transduction antagonist mediated by an EGFR family member and is now resistant to treatment with the antagonist. In some embodiments, the cancer of the subject to be treated relapses and/or progresses after treatment with a signaling antagonist mediated by an EGFR family member. In some embodiments, the cancer of the subject to be treated initially responds to treatment with a signaling antagonist mediated by an EGFR family member, but then progresses over said treatment.

In some embodiments, a subject to be treated according to the present invention may have been determined to have (i.e. may have been diagnosed with) a cancer comprising cells having a mutation that results in increased expression of HER3 ligand (e.g. as described herein). In some embodiments, the methods of the invention may comprise determining whether a subject has a cancer comprising a cell having a mutation that results in increased expression of HER3 ligand. In some embodiments, the method comprises analyzing nucleic acids from cancer cells. In some embodiments, the method comprises detecting a mutation that causes increased expression of HER3 ligand.

The skilled artisan readily determines the cancers and subjects described herein. Such cancers and subjects can be identified, for example, by monitoring the development/progression of the cancer (and/or its association) over a period of time, e.g., during treatment with an EGFR family member-mediated signaling antagonist. In some embodiments, identification of such subjects/cancers may include, for example, in vitro analysis (e.g., biopsy) of the sample. In some embodiments, cancers comprising associated mutant cells with reduced sensitivity and/or resistance to antagonist therapy may be identified. In some embodiments, a cancer comprising cells with upregulated expression of an EGFR family member can be identified.

In particular embodiments, the cancer to be treated is resistant to treatment with an antagonist of EGFR and/or HER 2-mediated signaling. In some embodiments, the cancer of the subject to be treated is resistant to treatment with an antagonist of EGFR and/or HER 2-mediated signaling. In some embodiments, the cancer of the subject to be treated has developed resistance to treatment with an EGFR and/or HER 2-mediated signaling antagonist. In some embodiments, the cancer of the subject to be treated has previously responded to treatment with an antagonist of signaling mediated by EGFR and/or HER2 and is now resistant to treatment with the antagonist. In some embodiments, the cancer of the subject to be treated relapses and/or progresses after treatment with an EGFR and/or HER 2-mediated signaling antagonist. In some embodiments, the cancer of the subject to be treated initially responds to treatment with an antagonist of signaling mediated by EGFR and/or HER2, but then progresses over the treatment.

In particular embodiments, the cancer to be treated comprises a mutation that is resistant to treatment with a BRAF inhibitor. In some embodiments, the mutation is a mutation of BRAF V600. In some embodiments, the mutation is BRAF V600E or V600K.

In particular embodiments, the cancer to be treated comprises a mutation that confers resistance to BRAF inhibitor therapy (e.g., a mutation at BRAF V600), and the therapy comprises administration of vemurafenib (vemurafenib) or dabrafenib (darafenib).

In some embodiments, the antigen binding molecule is administered in combination with an agent capable of inhibiting signaling mediated by the immune checkpoint molecule. In some embodiments, the immune checkpoint molecule is: such as PD-1, CTLA-4, LAG-3, VISTA, TIM-3, TIGIT or BTLA. In some embodiments, the antigen binding molecule is administered in combination with an agent capable of promoting signaling mediated by a co-stimulatory receptor. In some embodiments, the co-stimulatory receptor is: such as CD28, CD80, CD40L, CD86, OX40, 4-1BB, CD27, or ICOS.

Accordingly, the invention provides a composition comprising an article of manufacture of the invention (e.g., an antigen binding molecule of the invention) and an agent capable of inhibiting signaling mediated by an immune checkpoint molecule. Also provided are compositions comprising an article of manufacture of the invention and an agent capable of promoting signaling mediated by a co-stimulatory receptor. Also provided are uses of such compositions in the methods of medical treatment and prevention of diseases/disorders described herein.

Also provided are methods for treating/preventing a disease/disorder described herein, comprising administering an article of manufacture of the invention (e.g., an antigen binding molecule of the invention), and an agent capable of inhibiting signaling mediated by an immune checkpoint molecule. Also provided are methods for treating/preventing a disease/disorder described herein, comprising administering an article of manufacture of the invention (e.g., an antigen binding molecule of the invention), and an agent capable of inhibiting signaling mediated by a co-stimulatory receptor.

Agents capable of inhibiting signaling mediated by immune checkpoint molecules are known in the art and include: for example an antibody capable of binding to an immune checkpoint molecule or a ligand thereof and inhibiting immune checkpoint molecule mediated signalling. Other agents capable of inhibiting signaling mediated by an immune checkpoint molecule include agents capable of reducing gene/protein expression of the immune checkpoint molecule or a ligand of the immune checkpoint molecule (e.g., by inhibiting transcription of the gene encoding the immune checkpoint molecule/ligand, inhibiting post-transcriptional processing of RNA encoding the immune checkpoint molecule/ligand, reducing stability of RNA encoding the immune checkpoint molecule/ligand, promoting degradation of RNA encoding the immune checkpoint molecule/ligand, inhibiting post-translational processing of the immune checkpoint molecule/ligand, reducing stability of the immune checkpoint molecule/ligand, or promoting degradation of the immune checkpoint molecule/ligand) and small molecule inhibitors.

Agents capable of promoting signal transduction mediated by co-stimulatory receptors are known in the art and include, for example: agonist antibodies capable of binding to a co-stimulatory receptor and triggering or increasing signaling mediated by the co-stimulatory receptor. Other agents capable of promoting co-stimulatory receptor-mediated signaling include agents capable of increasing gene/protein expression of a co-stimulatory receptor or a ligand of a co-stimulatory receptor (e.g., by promoting transcription of a gene of a co-stimulatory receptor/ligand, promoting post-transcriptional processing of an RNA encoding a co-stimulatory receptor/ligand, increasing stability of an RNA encoding a co-stimulatory receptor/ligand, inhibiting degradation of an RNA encoding a co-stimulatory receptor/ligand, promoting post-translational processing of a co-stimulatory receptor/ligand, increasing stability of a co-stimulatory receptor/ligand, or inhibiting degradation of a co-stimulatory receptor/ligand) and small molecule inhibitors.

In particular embodiments, the antigen binding molecules of the present invention are administered in combination with an agent capable of inhibiting signaling mediated by PD-1. The agent capable of inhibiting signaling mediated by PD-1 may be an agent that targets PD-1 or PD-L1. The agent capable of inhibiting signal transduction mediated by PD-1 can be, for example, an antibody capable of binding to PD-1 or PD-L1 and inhibiting PD-1 mediated signal transduction.

In some embodiments, the antigen binding molecules of the present invention are administered in combination with an agent capable of inhibiting CTLA-4-mediated signaling. The agent capable of inhibiting CTLA-4-mediated signaling can be an agent that targets CTLA-4, or an agent that targets a ligand (e.g., CD80 or CD86) directed against CTLA-4. In some embodiments, the agent capable of inhibiting CTLA-4-mediated signaling can be, for example, an antibody capable of binding CTLA-4, CD80, or CD86 and inhibiting CTLA-4-mediated signaling.

In some embodiments, the antigen binding molecules of the present invention are administered in combination with an agent capable of inhibiting LAG-3 mediated signaling. The agent capable of inhibiting LAG-3 mediated signal transduction may be an agent that targets LAG-3, or may be an agent that targets LAG-3 ligands (e.g., MHC class II). In some embodiments, the agent capable of inhibiting LAG-3 mediated signaling can be, for example, an antibody capable of binding to LAG-3 or MHC class II and inhibiting LAG-3 mediated signaling.

In some embodiments, the antigen binding molecules of the present invention are administered in combination with an agent capable of inhibiting VISTA-mediated signaling. The agent capable of inhibiting VISTA-mediated signaling can be an agent that targets VISTA, or an agent that targets a VISTA ligand (e.g., VSIG-3 or VSIG-8). In some embodiments, the agent capable of inhibiting VISTA-mediated signaling can be, for example, an antibody capable of binding VISTA, VSIG-3 or VSIG-8 and inhibiting VISTA-mediated signaling.

In some embodiments, the antigen binding molecules of the present invention can be administered in combination with an agent that inhibits TIM-3 mediated signaling. The agent capable of inhibiting TIM-3 mediated signaling can be a TIM-3 targeting agent, or an agent directed against a TIM-3 ligand (e.g., Galectin 9). In some embodiments, an agent capable of inhibiting TIM-3 mediated signaling can be, for example, an antibody capable of binding to TIM-3 or Galectin 9 and inhibiting TIM-3 mediated signaling.

In some embodiments, the antigen binding molecules of the present invention are administered in combination with an agent capable of inhibiting TIGIT-mediated signaling. An agent capable of inhibiting TIGIT-mediated signal transduction may be an agent that targets TIGIT, or an agent that targets a TIGIT ligand (e.g., CD113, CD112, or CD 155). In some embodiments, the agent capable of inhibiting TIGIT-mediated signaling can be, for example, an antibody capable of binding to TIGIT, CD113, CD112, or CD155 and inhibiting TIGIT-mediated signaling.

In some embodiments, the antigen binding molecules of the present invention are administered in combination with an agent capable of inhibiting BTLA-mediated signaling. The agent capable of inhibiting BTLA-mediated signaling can be an agent that targets BTLA, or an agent that targets a ligand of BTLA (e.g., HVEM). In some embodiments, the agent capable of inhibiting BTLA-mediated signaling can be, for example, an antibody capable of binding to BTLA or HVEM and inhibiting BTLA-mediated signaling.

In some embodiments, the methods of using the antigen binding molecules of the invention in combination with agents capable of inhibiting signaling mediated by an immune checkpoint molecule (e.g., PD-1) provide improved therapeutic efficacy compared to the effects observed when one of the drugs is used as a monotherapy. In some embodiments, the combination of the antigen binding molecules of the invention and an agent capable of inhibiting signaling mediated by an immune checkpoint molecule (e.g., PD-1) provides a synergistic (i.e., superadditive) therapeutic effect.

By co-administration is meant that the antigen binding molecule, polypeptide, CAR, nucleic acid (or nucleic acids), expression vector (or expression vectors), cell, or composition is administered with the therapeutic agent, e.g., as a pharmaceutical composition comprising both drugs (combined preparation), or next to each other and optionally through the same route of administration, e.g., the same artery/vein or other blood vessel. Sequential administration refers to administration of one of the antigen binding molecule/composition or the therapeutic agent followed by administration of the other agent separately after a given time interval. Although this is the case in certain embodiments, it is not necessary that both agents be administered by the same route. The time interval may be any time interval.

Chemotherapy and radiotherapy refer to the treatment of cancer with drugs or ionizing radiation (e.g., radiotherapy using X-rays or gamma rays), respectively. The drug may be a chemical entity, such as a small molecule drug, an antibiotic, a DNA intercalator, a protein inhibitor (e.g. a kinase inhibitor) or a biological agent, such as an antibody, an antibody fragment, an aptamer, a nucleic acid (e.g. DNA, RNA), a peptide, a polypeptide or a protein. The medicament may be formulated as a pharmaceutical composition or medicament. The formulations may comprise one or more drugs (e.g. one or more active agents) together with one or more pharmaceutically acceptable diluents, excipients or carriers.

Treatment may involve the administration of more than one drug. Depending on the condition to be treated, the agents may be administered alone or in combination with other therapies, either simultaneously or sequentially. For example, chemotherapy may be a combination therapy involving the administration of two drugs, one or more of which may be intended to treat cancer.

Chemotherapy may be administered by one or more routes of administration, e.g., parenteral, intravenous injection, oral, subcutaneous, intradermal, or intratumoral.

Chemotherapy may be performed according to a treatment regimen. The treatment regimen may be a predetermined schedule, plan, protocol or schedule of chemotherapy administration, may be prepared by a physician or medical practitioner, and may be specifically tailored to suit the patient in need of treatment. The treatment regimen may indicate one or more of: the type of chemotherapy administered to the patient; the dose of each drug or radiation; the time interval between administrations; the time of each treatment; the number and nature (if any) of any treatment holidays. For combination therapy, a single treatment regimen may be provided that indicates how each drug is to be administered.

The chemotherapeutic agent may be selected from: abixet, abiraterone acetate, abexet (methotrexate), abexeson (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE-PC, AC, acatinib, AC-T, akriesi (vebuxib mab injection), ADE, trastuzumab-maytansine conjugate, doxorubicin (doxorubicin hydrochloride), afatinib dimaleate, femitory (everolimus), akuaze (netupitant and palonosetron hydrochloride), idamole (imiquimod), aldesleukin, ilexate (ellitinib), elotinib, alemtuzumab, tetai (disodium pemetrexenatide), alisib (palonosetron hydrochloride), icotlam (melphalan hydrochloride), ecliptan for injection (melphalan hydrochloride), ecliptam tablet (melphalan), alloxazine (palonosetron hydrochloride), aliskiren (palonosetron hydrochloride), argenz (bageri) and gefinitib (bagentine hydrochloride), Ambroxacin (chloramine), ampicillin (chloramine), amifostine, aminolevulinic acid, anastrozole, aprepitant, alexidine (disodium pamidronate), alimidide (anastrozole), arnotene (exemestane), alafenadine (nelarabine), arsenic trioxide, azinam (ofatumumab), asparaginase chrysanthemi pythium, argzolirtizumab, avastin (bevacizumab), avilamumab, yerkata, axitinib, azacitidine, venbaximo (avilamumab), beopapp, bescenu (nitrosourea mustard), belida (belinostat), belinostat, bendamustine hydrochloride, BEP, bevacizumab (otuzumab), bevacizumab, bexarotene (tositumomab and iodine I131 tositumomab), bicalutamide, bicucrium (bicucu) nitrosylurea, bevacizumab (nitrosylm, bevacizumab), bevacizumab (bevacizumab), bexat, bexarotene (bexat), bexat (bexatuzumab and iodine I131) Bleomycin, bortezomib (bosutinib), boscalid (bosutinib), bosutinib, vebuxib, bririgonib, BuMel, busulfan injection (busulfan), cabazitaxel, carboplatin (caboztinib malate), cabozantinib malate, CAF, carkuns (akaneib), campsis (alemtuzumab), kaposi (irinotecan hydrochloride), capecitabine, CAPOX, cararac (fluorouracil-topical), carboplatin-tacrolic, carfilzomib, carmobrevix (carmustine), carmustine implant, compactin (bicalutamide), CEM, retinib, cefuroxime (daunorubicin hydrochloride), valricekinuclidine (HPV vaccine), recombinant HPV (HPV v), cetuximab (cevax), Cex (CEM), cex (cevaglicotimib) Phenylbenzbicifloximane, CHOP, cisplatin, cladribine, clarithrone (cyclophosphamide), clofarabine (clofarabine), clolorane (clofarabine), CMF, cometinib, coumarone (cabozantinib malate), palboceprazole hydrochloride, COPDAC, COPP-ABV, dactinomycin (actinomycin), cotirib (cobicistinib), crizotinib, CVP, cyclophosphamide, Cyfos (ifosfamide), selazasa (ramucimaumab), cytarabine, Cytosar-U (cytarabine), carceracin (cyclophosphamide), dacrafenib, dacarbazine (decitabine), actinomycin, darunavir, dacramucirumab, megacolloxan (darumab), dasatinib, daunorubicin hydrochloride, and cytarabine, decitabine, defibroside, descellavine sodium, descellulan sodium (sodium), delibrinorin (sodium D) Degarelix, dinierein, dinolizumab, dexrazoxane, dinotefuran, doxetaxel, Doxil, epirubicin hydrochloride, doxorubicin hydrochloride, Dox-SL (doxorubicin hydrochloride liposome), DTIC-doxme (dacarbazine), doluzumab, Efudex (fluorouracil-topical), erilast (labisidase), epirubicin (epirubicin hydrochloride), elobizumab, eloxatin (oxaliplatin), eltrombopag, merepirubicin (aprepirubicin), eliximab, eloxatin (oxaliplatin), eltamitocin, rilipine (aprepirubicin), eloxib (elobizumab), enidolipine mesylate, lenamide, epirubicin hydrochloride, EPOCH, etoricombicib (cetuximab), eprinomycin mesylate, riligy (vismodex), eigine hydrochloride, ezetimiun (asparaginase), eremophila, Ethyol (amifostine), etoposide phosphate (etoposide phosphate), etoposide phosphate, efacil (doxorubicin HCl liposome), everolimus, everitamin (raloxifene HCl), efamelat (melphalan HCl), exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil-topical), fareton (toremifene), fadak (panobistat), fude (fluvistine), FEC, Freon (letrozole), feglastin, fludara (fludarabine phosphate), fludarabine phosphate, fluoro complex (fluorouracil-topical), fluorouracil injection, fluorouracil-topical, flutamide, Folex (methotrexate), Folex PFS (methotrexate), Foerflies, Furfeiri, Furfeili-bevacizumab, Foleflutamb, Forferi-cetuximab, Forferilin, Folfrostris, Folotting (an acetyl carbamate), FU-LV, fluvistron, Gardney (a recombinant HPV tetravalent vaccine), Gardney 9 (a recombinant HPV nine-valent vaccine), Gardney (an Obbinomizumab), Gefitinib, Gemcitabine-Cisabamine hydrochloride, Gemcitabine-oxaliplatin, Getuzumab geutib, Gemma (Gemcitabine hydrochloride), Gelotritrevif (Afatinib dimaleate), Gleevec (Imatinib mesylate), Gelididel (a Carmositin implant), Grignard wafer (a Carmusitin implant), carboxypeptidase, Gotherlin acetate, Han (Ellipulin mesylate), Herman (propranolol hydrochloride), herceptin (trastuzumab), a bivalent HPV vaccine, recombinant, Hermangol (a hydrochloride), Herceptin (a trastuzumab), and a vaccine, HPV nine vaccine, recombinant, HPV tetravalent vaccine, recombinant and mefenacin (topotecan hydrochloride), hydroxyurea (hydroxyurea), hydroxyurea, Hyper-CVAD, Ebosin (palbociclib), iburitumab, ibrutinib, ICE, Ikruex (ponatinib hydrochloride), idamycin (idarubicin hydrochloride), idarubicin hydrochloride, idalisib, Edisonib mesylate, ifferenil (ifosfamide), ifosfamide (ifosfamide), IL-2 (aldesleukin), imatinib mesylate, ibuvicat (ibrutinib), efenii (doluzumab), imiquimod, ligosacce (oncolytic virus), rituximab (axinib), etolizumab, interferon Alfa-2b, recombinant interleukin 2 (aldesleukin), intron A (interferon Alfa-2b), recombinant Alfa-2b), interferon Alfa-2b, recombinant interleukin, Iodine I131 tositumumab and tositumumab, ipilimumab, iressa (gefitinib), irinotecan hydrochloride liposome, ibritumomab (romidepsin), ixabepilone, elsamizolamide citrate, ixabepilone, giardia (ruxolitinib phosphate), JEB, yeritana (cabazitaxel), casira (trastuzumab-maytansine conjugate), ketorolfine (raloxifene hydrochloride), kepidium (palivimine), katemlub (pembrolizumab), cisapride (resilica), aurigo (tirapanib), chrysia (tikovia nucleus), cheopipris (carfilzomib), lanreotide acetate, lapatinib ditosylate, larrufoucaulumab (olanzab), len, lenalidomide, mervatinib mesylate, rivastigmine (mesylate), letrovulanib, letrozole, calcium butyrate, clenbuterol (clenbuterol), clenbuterol (clotrimazole), valacil (clocrine), valacil (clocrine), gefitinib (clorfl) and gawarfarland (clorfl) hydrochloride (clorfl) liposome-n-b-d-b-n-c acid, Leuprolide acetate, lovastatin (cladribine), reuptamine (aminolevulinic acid), rifamine (chlorobutyric acid), lipdol (doxorubicin liposome hydrochloride), lomustine, Langerv (trifluoropyridine and tipiracil hydrochloride), lupulon (leuprolide acetate), long-acting lupulon (leuprolide acetate), Linpazae (Oriparib), mazopu (vincristine sulfate liposome), Mandarin (carbamazine hydrochloride), propisochlor hydrochloride, pregnenolone acetate, Mekinsonite (triamcinolone), Melphalan hydrochloride, mercaptopurine, Meissner (Meissner), metrazone (temozolomide), methotrexate (methotrexate), bromomethylnaltrexone, Mekratrexate (methotrexate), Mekratrexate-LPF-AQ (methotrexate), Medullosin, mitomycin C, mitoxantrone hydrochloride, mitochondrial aspergillin (mitomycin C), MOPP, mozabi (pelizaifu), sinapine (methoxyethylamine hydrochloride), mutamycin (mitomycin C), Millerlan (busulfan), Melarosal (azacitidine), Milotag (gemtuzumab ozogamicin), nanoparticulate paclitaxel (paclitaxel albumin-stabilized nanoparticulate formulation), Nanweibine (vinorelbine tartrate), Nibizumab, Nelarabine, Niosal (cyclophosphamide), maleic acid, Liss (Nelartinib maleate), Nepitant and Palonosetron hydrochloride, Nolatta (pegfilgrastim), Nepetin (Fegrastimastin), Leisha (Sorafeniumtoluene sulfonate), Nilandellolone (Ninitanide), Ninitonie, nilutamide (citric acid, nilutamide), nilutamide, and nilutamide, Nilapalbumin tosylate monohydrate, nivoruzumab, norvedol (tamoxifen citrate), naltrexate (romidepsin), obinuotuzumab, ondozolomide (sondoxib), OEPA, otatuzumab, OFF, olaparib, olamab, omaxetine mesylate succinate, encapsa (pegatrizyme), ondansetron hydrochloride, ornivide (irinotecan hydrochloride liposome), otake (dinleukin), opropyl (nivolumab), OPPA, ocitinib, oxaliplatin, paclitaxel albumin-stabilized nanoparticle formulations, PAD, pabuxib, palifermin, palonosetron hydrochloride, and endopitant, disodium pamidronate, panitumumab, palonobetasol, cisplatin (carboplatin), carboplatin hydrochloride, pazoplatin), zoledrine (carboplatin), zoledrine (carvaclatin hydrochloride), zoledrine (zoledrine), ritin hydrochloride, and endoprostatin, PCV, PEB, pemetrexed, pirfenitin, peginterferon Alfa-2b, PEG-intron (PEG interferon Alfa-2b), pemetrexed disodium, peltata (pertuzumab), pertuzumab, platinol-AQ (cisplatin), pellucaflavine, pomalidomide, pommalidomide (pomalidomide), pranatine hydrochloride, bordeaux (nimotuzumab), prasugrec acid, prednisone, procarbazine hydrochloride, propleukin (aldesleukin), pulolinia (dinomab), pralata (elta), propranolab (elta), propranolol hydrochloride, propamocarb (sipelol), rinite (mercaptopurine), pregabar (mercaptopurine), [ nonet ] 223 dichloride, raloxifene hydrochloride, raloximab, mustard, R-CHOP, R-CVP, recombinant Human Papilloma Virus (HPV) bivalent vaccine, recombinant Human Papilloma Virus (HPV) nine-valent vaccine, recombinant Human Papilloma Virus (HPV) tetravalent vaccine, recombinant interferon Alfa-2b, Ragofenib, oral methylnaltrexone bromide (methylnaltrexone bromide), R-EPOCH, Redumet (lenalidomide), Remateiral (methotrexate), Ribosexide, R-ICE, Rituxan (Rituximab), Rituxan (Rituximab and human hyaluronidase), Rituximab and human hyaluronidase, Lapidan hydrochloride, Romidepsine, Romidepstein, daunorubicin hydrochloride, daunomycin (daunorubicin hydrochloride), Ribazakhapra camphorsulfonate, Ruxolitinib phosphate, Ridada (Soundurin), Sterlan pleuropneumoniae (Talc powder), Cetuximab, cetrapel, long-acting somatotropin (lanreotide acetate), sonedgi, sorafenib tosylate, prilisib (dasatinib), stanford V, sterile talc (talc), staurtick (talc), stuvagal (ragoldney), sunitinib malate, suttan (sunitinib malate), saladrolone (PEG interferon Alfa-2b), cetuximab (cetuximab), trilibat (omeprazole hydrochloride), tebuconazole (thioguanine), TAC, tafelodil (dalanib), tagesol (axitinib), talc, talimuranavirus, tamoxifen citrate, talabinfpfs (cytarabine), tarceva (erlotinib hydrochloride), tagagliptin (bexarotene), tacergine (nilotinib), paclitaxel (paclitaxel), and paclitaxel (paclitaxel) Taxotere (docetaxel), tesonique (arzolizumab), temozolomide (temozolomide), temozolomide, thalidomide (thalidomide), thioguanine, tiopagine, tirapa, Tiuma radicle, torac (fluorouracil-topical), topotecan hydrochloride, toremifene, torecyclamate (temsirolimus), tositumomab and iodo I131 tositumomab, tolmetin (dilazone hydrochloride), TPF, trabedine, trimetinib, trastuzumab, tranexamida (bendamustine hydrochloride), trazodistan (bendamustine hydrochloride), trifluridine hydrochloride/tipepidine, trascinonide (arsenic trioxide), tebucarb (lapatinib xylene sulfonate), neoxizumab (dexrazumab), uridine triacetate, VAC, valrubicin, valacilin (valfloxacin), valvactilrubicin, valtretafloxacin, VAMP, ruvapitab (hydrochloride), Vicotinib (panitumumab), VeIP, vepitot (vinblastine sulfate), velcade (bortezomib), vilsa (vinblastine sulfate), vilafenib, vinitox (vinitox sulfate), vinitox, wellenio (Abemascib), vilde (leuprolide acetate), vildagarel (azacitidine), vinblastine sulfate, vinxsapos (vincristine sulfate), vincristine sulfate liposomes, vinorelbine tartrate, VIP, Virgimod, Vistogarde (uridine triacetate), voraxane (glucosidase), ritotat, vorexane (Pazopanib hydrochloride), vex (dactinomycin hydrochloride and cytarabine hydrochloride liposomes), vesuvix (levosovix hydrochloride calcium), viccovarel (krusettuytinib), rhodarabine (capecitabine), laistacitabine, surimib, schlepigler, velvetivorelbine, valtrexate, valtrexase hydrochloride, Denosumab (dinomab), kexophenanthrol (radium dichloride 223), custans (enzalutamide), jeffer (ipilimumab), yerkata (aici), sulinds (telabetin), zatript (aflibercept), zalcicept (feigastine), zerewitinola (nipagoda monohydrate tosylate), zelboraf (vilafinil), zedrolin (ibularmab), neocado (dilazone hydrochloride), aflibercept, zoffia (ondansetron hydrochloride), norrad (sarerelin acetate), zoledronic acid, zollinga (vorinostat), zoledronic acid (zoledram), zafirarnica (clidiniazide acetate), and abicadia (abiraterone acetate).

In some embodiments, the antigen binding molecules of the invention are administered in combination with one or more of trastuzumab, cetuximab, cisplatin, 5-FU, or capecitabine. In some embodiments, the antigen binding molecules of the present invention are administered in combination with trastuzumab and cisplatin, and 5-FU or capecitabine.

In some embodiments, the antigen binding molecules of the present invention are administered in combination with cetuximab. Administration in combination with cetuximab is particularly contemplated for the treatment of head and neck cancer (e.g., head and neck squamous cell carcinoma).

Multiple doses of the antigen binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell, or composition may be provided. One or more doses or each dose may be concomitant with the simultaneous or sequential administration of another therapeutic agent.

The plurality of doses may be separated by a predetermined time interval, which may be selected to be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or one of 1, 2, 3, 4, 5, or 6 months. For example, a dose may be administered every 7, 14, 21 or 28 days (plus or

minus

3, 2 or 1 day).

Detection method

The invention also provides an article of manufacture of the invention for use in a method of detecting, localizing or imaging HER3 or a cell expressing HER 3.

The antigen binding molecules described herein may be used in methods involving an antigen binding molecule of HER 3. Such methods may involve detecting a binding complex of an antigen binding molecule and HER 3.

As such, methods are provided that include contacting a sample comprising or suspected of comprising HER3 and detecting the formation of a complex of an antigen binding molecule and HER 3. Also provided are methods comprising contacting a sample comprising or suspected of comprising a cell expressing HER3 and detecting the formation of a complex of an antigen binding molecule and a cell expressing HER 3.

Suitable formats of methods are well known in the art and include immunoassays, for example sandwich assays, such as ELISA. The method may comprise labeling the antigen binding molecule or target, or both, with a detectable moiety, such as a fluorescent label, phosphorescent label, luminescent label, immunologically detectable label, radioactive label, chemical, nucleic acid, or enzymatic label as described herein. Detection techniques are well known to those skilled in the art and may be selected to correspond to the labeling agent.

Such methods may provide a basis for diagnostic or/and prognostic assessment methods for diseases or disorders, such as cancer. Such methods may be performed on patient samples in vitro, or after treatment of patient samples. Once the sample is collected, there is no need for the patient to be present in an in vitro method to be performed, and thus the method may be a method which is not practiced on the human or animal body. In some embodiments, the method is performed in vivo.

Detection in a sample can be used for the purpose of diagnosing a disease/disorder (e.g., cancer), a susceptibility to a disease/disorder, or to provide a prognosis (prediction) of a disease/disorder, e.g., a disease/disorder described herein. The diagnosis or prognosis may be related to an existing (previously diagnosed) disease/disorder.

Such methods may involve detecting or quantifying HER3 or HER3 expressing cells in a patient sample, for example. If the method includes quantifying the relevant factor, the method may further include comparing the measured amount to a standard or reference value as part of a diagnostic or prognostic assessment. Other diagnostic/prognostic tests can be used in conjunction with the tests described herein to increase the accuracy of the diagnosis or prognosis or to confirm the results obtained using the tests described herein.

The sample may be obtained from any tissue or body fluid. The sample may comprise or may be derived from: a quantity of blood; an amount of serum derived from the blood of an individual, possibly comprising a liquid fraction of the blood obtained after removal of fibrin clots and blood cells; a tissue sample or biopsy sample; hydrothorax; cerebrospinal fluid (CSF); or a cell isolated from the individual. In some embodiments, the sample may be obtained or derived from one or more tissues affected by the disease/disorder (e.g., one or more tissues in which disease symptoms are present or the pathogenesis of the disease/disorder is involved).

The invention also provides methods of selecting/ranking subjects for treatment with HER3 targeting agents. In some embodiments, a subject is selected for a treatment/prevention method of the invention, or identified as a subject that would benefit from such a treatment/prevention method, based on the detection/quantification of HER3 or HER 3-expressing cells in a sample obtained, e.g., from the subject.

Object

According to aspects of the invention described herein, the subject may be any animal or human. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but more preferably is a human. The subject may be male or female. The subject may be a patient. A subject may have been diagnosed with a disease or condition requiring treatment (e.g., cancer), may be suspected of having such a disease/condition, or may be at risk of developing/having such a disease/condition.

In an embodiment of the invention, the subject is preferably a human subject. In some embodiments, the subject to be treated by the therapeutic or prophylactic methods of the invention is a subject having or at risk of having cancer. In embodiments of the invention, a subject may be selected for treatment according to the method based on the characterization of certain markers of such diseases/disorders.

Reagent kit

In some aspects of the invention described herein, kits are provided in parts. In some embodiments, a kit can have at least one container with a predetermined amount of an antigen binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell, or composition described herein.

In some embodiments, a kit can comprise materials for producing an antigen binding molecule, polypeptide, CAR, nucleic acid (or a plurality thereof), expression vector (or a plurality thereof), cell, or composition described herein.

The kit can provide an antigen binding molecule, polypeptide, CAR, nucleic acid (or a plurality thereof), expression vector (or a plurality thereof), cell, or composition, and instructions for administering to a patient to treat a particular disease/disorder.

In some embodiments, the kit may further comprise at least one container having a predetermined amount of another therapeutic agent (e.g., an anti-infective agent or a chemotherapeutic agent). In such embodiments, the kit may further comprise a second drug or pharmaceutical composition such that both drugs or pharmaceutical compositions may be administered simultaneously or separately, thereby providing a combination therapy for a particular disease or condition. The therapeutic agent may also be formulated to be suitable for injection or infusion into the tumor or blood.

Sequence identity

As used herein, "sequence identity" refers to the percentage of nucleotides/amino acid residues in the subject sequence that are identical to the nucleotides/amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity between the sequences. To determine the percent sequence identity between two or more amino acid or nucleic acid sequences, pairwise and multiple sequence alignments can be accomplished in a variety of ways known to those skilled in the art, for example, using publicly available computer software, such as Clustalomega

J., 2005, Bioinformatics 21, 951-. When such software is used, it is preferred to use default parameters such as gap penalties and extension penalties.

Sequence of

Numbered paragraphs

The following numbered paragraphs (paragraphs) provide further statements of features and combinations of features contemplated as being relevant to the present invention:

1. optionally an isolated antigen binding molecule capable of binding to HER3 in extracellular domain subdomain II.

2. The antigen binding molecule of paragraph 1, wherein said antigen binding molecule inhibits the interaction between HER3 and HER3 interacting partners.

3. The antigen binding molecule of

paragraph

1 or 2, wherein said antigen binding molecule is capable of binding to a polypeptide comprising or consisting of SEQ ID NO: 16.

4. The antigen binding molecule of any one of paragraphs 1 to 3, wherein said antigen binding molecule is capable of binding to a polypeptide comprising SEQ ID NO: 23 or SEQ ID NO: 229.

5. The antigen binding molecule of any one of paragraphs 1 to 4, wherein said antigen binding molecule is capable of binding to a polypeptide comprising SEQ ID NO: 21 or SEQ ID NO: 229.

6. The antigen binding molecule of any one of paragraphs 1 to 5, wherein the antigen binding molecule comprises:

(i) a heavy chain variable region (VH) comprising the following CDRs:

HC-CDR2 having amino acid sequence shown in SEQ ID NO. 46

Has the sequence of SEQ ID NO: 51, HC-CDR 3; and

(ii) a light chain variable region (VL) comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 91

LC-CDR2 having the amino acid sequence shown in SEQ ID NO 94

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 99.

7. The antigen binding molecule of any one of paragraphs 1 to 6, wherein the antigen binding molecule comprises:

(i) a heavy chain variable region (VH) comprising the following CDRs:

HC-CDR1 having the amino acid sequence shown in SEQ ID NO. 41

HC-CDR2 having the amino acid sequence shown in SEQ ID NO. 44

HC-CDR3 having the amino acid sequence set forth in SEQ ID NO. 47; and

(ii) a light chain variable region (VL) comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 88

LC-CDR2 having the amino acid sequence shown in SEQ ID NO. 92

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 95.

8. The antigen binding molecule of any one of paragraphs 1 to 6, wherein the antigen binding molecule comprises:

(i) a heavy chain variable region (VH) comprising the following CDRs:

HC-CDR1 having amino acid sequence shown in SEQ ID NO. 41

HC-CDR2 having amino acid sequence shown in SEQ ID NO. 44

HC-CDR3 having the amino acid sequence set forth in SEQ ID NO. 47; and

(ii) a light chain variable region (VL) comprising the following CDRs:

has the sequence of SEQ ID NO: 89, or an LC-CDR1 of the amino acid sequence shown in

LC-CDR2 having the amino acid sequence shown in SEQ ID NO. 92

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 95.

9. The antigen binding molecule of any one of paragraphs 1 to 6, wherein the antigen binding molecule comprises:

(i) a heavy chain variable region (VH) comprising the following CDRs:

HC-CDR1 having the amino acid sequence shown in SEQ ID NO. 41

HC-CDR2 having the amino acid sequence shown in SEQ ID NO. 44

HC-CDR3 having the amino acid sequence set forth in SEQ ID NO. 47; and

(ii) a light chain variable region (VL) comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 90

LC-CDR2 having the amino acid sequence shown in SEQ ID NO. 92

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 96.

10. The antigen binding molecule of any one of paragraphs 1 to 6, wherein the antigen binding molecule comprises:

(i) a heavy chain variable region (VH) comprising the following CDRs:

HC-CDR1 having the amino acid sequence shown in SEQ ID NO. 41

HC-CDR2 having amino acid sequence shown in SEQ ID NO. 44

HC-CDR3 having the amino acid sequence set forth in SEQ ID NO. 47; and

(ii) a light chain variable region (VL) comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 88

LC-CDR2 having the amino acid sequence shown in SEQ ID NO. 92

An LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 98.

11. The antigen binding molecule of any one of paragraphs 1 to 6, wherein the antigen binding molecule comprises:

(i) a heavy chain variable region (VH) comprising the following CDRs:

HC-CDR1 having the amino acid sequence shown in SEQ ID NO. 41

HC-CDR2 having the amino acid sequence shown in SEQ ID NO. 45

HC-CDR3 having the amino acid sequence set forth in SEQ ID NO. 47; and

(ii) a light chain variable region (VL) comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 88

LC-CDR2 having amino acid sequence shown in SEQ ID NO. 93

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 95.

12. The antigen binding molecule of any one of paragraphs 1 to 6, wherein the antigen binding molecule comprises:

(i) a heavy chain variable region (VH) comprising the following CDRs:

HC-CDR1 having amino acid sequence shown in SEQ ID NO. 41

HC-CDR2 having the amino acid sequence shown in SEQ ID NO. 45

HC-CDR3 having the amino acid sequence shown in SEQ ID NO. 49; and moreover

(ii) A light chain variable region (VL) comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 88

LC-CDR2 having the amino acid sequence shown in SEQ ID NO. 93

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 95.

13. The antigen binding molecule of any one of paragraphs 1 to 6, wherein the antigen binding molecule comprises:

(i) A heavy chain variable region (VH) comprising the following CDRs:

HC-CDR1 having the amino acid sequence shown in SEQ ID NO. 41

HC-CDR2 having the amino acid sequence shown in SEQ ID NO. 45

HC-CDR3 having the amino acid sequence shown in SEQ ID NO. 50; and

(ii) a light chain variable region (VL) comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 88

LC-CDR2 having the amino acid sequence shown in SEQ ID NO. 93

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 95.

14. The antigen binding molecule of any one of paragraphs 1 to 6, wherein the antigen binding molecule comprises:

(i) a heavy chain variable region (VH) comprising the following CDRs:

HC-CDR1 having the amino acid sequence shown in SEQ ID NO. 41

HC-CDR2 having the amino acid sequence shown in SEQ ID NO. 45

HC-CDR3 having the amino acid sequence shown in SEQ ID NO. 48; and

(ii) a light chain variable region (VL) comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 88

LC-CDR2 having the amino acid sequence shown in SEQ ID NO. 92

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 95.

15. The antigen binding molecule of any one of paragraphs 1 to 6, wherein the antigen binding molecule comprises:

(i) a heavy chain variable region (VH) comprising the following CDRs:

HC-CDR1 having the amino acid sequence shown in SEQ ID NO. 41

HC-CDR2 having the amino acid sequence shown in SEQ ID NO. 45

HC-CDR3 having the amino acid sequence shown in SEQ ID NO. 48; and

(ii) a light chain variable region (VL) comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 88

LC-CDR2 having the amino acid sequence shown in SEQ ID NO. 92

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 97.

16. The antigen binding molecule of any one of paragraphs 1 to 6, wherein the antigen binding molecule comprises:

(i) a heavy chain variable region (VH) comprising the following CDRs:

HC-CDR1 having the amino acid sequence shown in SEQ ID NO. 42

HC-CDR2 having the amino acid sequence shown in SEQ ID NO. 45

HC-CDR3 having the amino acid sequence shown in SEQ ID NO. 48; and

(ii) a light chain variable region (VL) comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 88

LC-CDR2 having the amino acid sequence shown in SEQ ID NO. 92

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 95.

17. The antigen binding molecule of any one of paragraphs 1 to 5, wherein the antigen binding molecule comprises:

(i) a heavy chain variable region (VH) comprising the following CDRs:

HC-CDR1 having amino acid sequence shown in SEQ ID NO. 158

HC-CDR2 having the amino acid sequence shown in SEQ ID NO. 159

HC-CDR3 having the amino acid sequence shown in SEQ ID NO. 160; and

(ii) a light chain variable region (VL) comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 165

LC-CDR2 having the amino acid sequence shown in SEQ ID NO 166

LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 167.

18. The antigen binding molecule of any one of paragraphs 1 to 4, wherein the antigen binding molecule is capable of binding to a polypeptide comprising EQ ID NO: 22.

19. The antigen binding molecule according to any one of paragraphs 1 to 4 or 18, wherein the antigen binding molecule comprises:

(i) a heavy chain variable region (VH) comprising the following CDRs:

HC-CDR1 having the amino acid sequence shown in SEQ ID NO. 128

HC-CDR2 having the amino acid sequence shown in SEQ ID NO. 129

HC-CDR3 having the amino acid sequence shown in SEQ ID NO. 130; and

(ii) a light chain variable region (VL) comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 136

LC-CDR2 having amino acid sequence shown in SEQ ID NO. 137

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 138.

20. The antigen binding molecule according to any one of paragraphs 1 to 4 or 18, wherein the antigen binding molecule comprises:

(i) A heavy chain variable region (VH) comprising the following CDRs:

HC-CDR1 having the amino acid sequence shown in SEQ ID NO 144

HC-CDR2 having the amino acid sequence shown in SEQ ID NO. 145

HC-CDR3 having the amino acid sequence shown in SEQ ID NO. 146; and

(ii) a light chain variable region (VL) comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 151

LC-CDR2 having the amino acid sequence shown in SEQ ID NO. 152

LC-CDR3 having the amino acid sequence shown in SEQ ID NO 153.

21. The antigen binding molecule of any one of paragraphs 1 to 4, wherein the antigen binding molecule comprises:

(i) a VH region comprising an amino acid sequence that is identical to SEQ ID NO: 24 has at least 70% sequence identity; and

a VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 74 has at least 70% sequence identity;

or

(ii) A VH region comprising an amino acid sequence identical to SEQ ID NO: 25 has at least 70% sequence identity; and

a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO 75;

or

(iii) A VH region comprising an amino acid sequence identical to SEQ ID NO: 26 has at least 70% sequence identity; and

A VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 76 has at least 70% sequence identity;

or

(iv) A VH region comprising an amino acid sequence identical to SEQ ID NO: 27 has at least 70% sequence identity; and

a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 77;

or

(v) A VH region comprising an amino acid sequence that is identical to SEQ ID NO: 28 has at least 70% sequence identity; and

a VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 78 has at least 70% sequence identity;

or

(vi) A VH region comprising an amino acid sequence that is identical to SEQ ID NO: 29 has at least 70% sequence identity; and

or

(vii) A VH region comprising an amino acid sequence that is identical to SEQ ID NO: 30 has at least 70% sequence identity; and

Or

(viii) A VH region comprising an amino acid sequence that is identical to SEQ ID NO: 31 has at least 70% sequence identity; and

a VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 79 have at least 70% sequence identity;

or

(ix) A VH region comprising an amino acid sequence that is identical to SEQ ID NO: 32 has at least 70% sequence identity; and

or

(x) A VH region comprising an amino acid sequence identical to SEQ ID NO: 33 has at least 70% sequence identity to the amino acid sequence set forth in seq id no; and

a VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 80 has at least 70% sequence identity;

or

(xi) A VH region comprising an amino acid sequence identical to SEQ ID NO: 34 has at least 70% sequence identity; and

a VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 81 has at least 70% sequence identity;

or

(xii) A VH region comprising an amino acid sequence that is identical to SEQ ID NO: 35 has at least 70% sequence identity; and

A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 82;

or

(xiii) A VH region comprising an amino acid sequence that is identical to SEQ ID NO: 36 has at least 70% sequence identity; and

a VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 83 has at least 70% sequence identity;

or

(xiv) A VH region comprising an amino acid sequence that is identical to SEQ ID NO: 37 has at least 70% sequence identity; and

a VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 84 has at least 70% sequence identity;

or

(xv) A VH region comprising an amino acid sequence identical to SEQ ID NO: 38 has at least 70% sequence identity; and

a VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 85 has at least 70% sequence identity;

or

(xvi) A VH region comprising an amino acid sequence identical to SEQ ID NO: 39 has at least 70% sequence identity; and

a VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 86 has at least 70% sequence identity;

Or

(xvii) A VH region comprising an amino acid sequence that is identical to SEQ ID NO: 40 has at least 70% sequence identity; and

a VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 87 has at least 70% sequence identity;

or

(xviii) A VH region comprising an amino acid sequence identical to SEQ ID NO: 127 has at least 70% sequence identity; and

a VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 135 has at least 70% sequence identity;

or

(xix) A VH region comprising an amino acid sequence identical to SEQ ID NO: 143 has at least 70% sequence identity; and

a VL region comprising an amino acid sequence that is substantially identical to SEQ ID NO: 150 has at least 70% sequence identity;

or

(xx) A VH region comprising an amino acid sequence that is identical to SEQ ID NO: 157 has at least 70% sequence identity; and

comprising a VL region having an amino acid sequence with at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 164.

22. The antigen binding molecule of any one of paragraphs 1 to 21, wherein the antigen binding molecule is capable of binding to human HER3 and one or more of mouse HER3, rat HER3 and cynomolgus monkey HER 3.

23. An optionally isolated antigen binding molecule comprising (i) an antigen binding molecule according to any one of paragraphs 1 to 22, and (ii) an antigen binding molecule capable of binding to an antigen other than HER 3.

24. The antigen binding molecule according to any one of paragraphs 1 to 23, wherein said antigen binding molecule is capable of binding to a cell that expresses HER3 on the surface of the cell.

25. The antigen binding molecule according to any one of paragraphs 1 to 24, wherein the antigen binding molecule is capable of inhibiting HER 3-mediated signal transduction.

26. The antigen binding molecule of any one of paragraphs 1 to 25, wherein the antigen binding molecule comprises an Fc region comprising a polypeptide having: (i) has C at a position corresponding to 242 bits and C at a position corresponding to 334 bits, and (ii) one or more of: there is a at the position corresponding to bit 236, D at the position corresponding to bit 239, E at the position corresponding to bit 332, L at the position corresponding to bit 330, K at the position corresponding to bit 345, and G at the position corresponding to bit 430.

27. The antigen binding molecule of paragraph 26, wherein the Fc region comprises a polypeptide having a C at the position corresponding to position 242, a C at the position corresponding to position 334, an a at the position corresponding to position 236, a D at the position corresponding to position 239, an E at the position corresponding to position 332, and an L at the position corresponding to position 330.

28. A Chimeric Antigen Receptor (CAR) comprising the antigen binding molecule of any one of paragraphs 1 to 27.

29. One or more optionally isolated nucleic acids encoding an antigen binding molecule according to any of paragraphs 1 to 27 or a CAR according to paragraph 28.

30. An expression vector or vectors comprising a nucleic acid or nucleic acids according to paragraph 29.

31. A cell comprising an antigen binding molecule according to any one of paragraphs 1 to 27, a CAR according to paragraph 28, a nucleic acid or nucleic acids according to paragraph 29, or an expression vector or expression vectors according to paragraph 29 through paragraph 30.

32. A method comprising culturing a cell comprising a nucleic acid or nucleic acids according to paragraph 29, or an expression vector or expression vectors according to paragraph 30, under conditions suitable for expression of an antigen binding molecule or CAR from the nucleic acid or nucleic acids or expression vector or vectors.

33. A composition comprising an antigen binding molecule according to any of paragraphs 1 to 27, a CAR according to paragraph 28, a nucleic acid or nucleic acids according to paragraph 29, an expression vector or vectors according to paragraph 30, or a cell of paragraph 31.

34. An antigen binding molecule according to any one of paragraphs 1 to 27, a CAR according to paragraph 28, one or more nucleic acids according to paragraph 29, an expression vector or vectors according to paragraph 30, a cell according to paragraph 31, or a composition according to paragraph 33 for use in a medical or prophylactic method.

35. An antigen binding molecule according to any one of paragraphs 1 to 27, a CAR according to paragraph 28, one or more nucleic acids according to paragraph 29, an expression vector or vectors according to paragraph 30, a cell according to paragraph 31 or a composition according to paragraph 33, for use in a method of treating or preventing cancer.

36. Use of an antigen binding molecule according to any one of paragraphs 1 to 27, a CAR according to paragraph 28, one or more nucleic acids according to paragraph 29, an expression vector or vectors according to paragraph 30, a cell according to paragraph 31 or a composition according to paragraph 33, for the manufacture of a medicament for use in a method of treating or preventing cancer.

37. A method of treating or preventing cancer, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen binding molecule according to any one of paragraphs 1 to 27, a CAR according to paragraph 28, a nucleic acid or nucleic acids according to paragraph 29, an expression vector or vectors according to paragraph 30, a cell of paragraph 31 or a composition of paragraph 33.

38. The antigen binding molecule, CAR, one or more nucleic acids, expression vector or vectors, cell or composition for use according to

paragraph

34 or 35, the use according to paragraph 36 or the method according to paragraph 37, wherein the method further comprises administering an inhibitor of signal transduction mediated by an EGFR family member, optionally wherein the inhibitor of signal transduction mediated by an EGFR family member is an inhibitor of signal transduction mediated by HER2 and/or EGFR.

39. The antigen binding molecule, CAR, nucleic acid or nucleic acids, expression vector or expression vectors, cell or composition of any one of paragraphs 34 to 38, wherein the cancer is selected from: cancer comprising cells expressing an EGFR family member, cancer comprising cells expressing HER3, solid tumors, breast cancer, malignant epithelial cancer of the breast, ductal carcinoma, gastric cancer, malignant epithelial cancer of the stomach, gastric adenocarcinoma, colorectal cancer, malignant epithelial cancer of the colorectal, colorectal adenocarcinomas, head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN), lung cancer, malignant epithelial cancer of the lung, squamous cell lung malignant epithelial cancer, ovarian cancer, malignant epithelial cancer of the ovary, ovarian serous adenocarcinoma, kidney cancer, malignant epithelial cancer of the kidney cell, clear cell cancer of the kidney, adenocarcinoma of the kidney cell, papillary cell cancer of the kidney, pancreatic cancer, adenocarcinoma of the pancreatic duct, cervical cancer, squamous cell carcinoma of the cervix, skin cancer, melanoma, esophageal carcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, uterine cancer, endometrial cancer, thyroid cancer, malignant epithelial cancer of the thyroid, chromophobe cancer, pheochromocytoma cancer, melanoma, pancreatic carcinoma, biliary tree carcinoma, pancreatic carcinoma, biliary tree carcinoma, pancreatic carcinoma, and/for example carcinoma, and/for the like, Paraganglioma, bladder cancer, bladder urothelial cancer, prostate adenocarcinoma, sarcoma, and thymoma.

40. A method of inhibiting HER 3-mediated signaling comprising contacting a cell expressing HER3 with an antigen binding molecule according to any one of paragraphs 1 to 27.

41. A method of reducing the number or activity of a cell expressing HER3, the method comprising contacting a cell expressing HER3 with an antigen binding molecule according to any one of paragraphs 1-27.

42. An optionally isolated in vitro complex comprising an antigen binding molecule according to any one of paragraphs 1 to 27 that binds to HER 3.

43. A method comprising contacting a sample containing or suspected of containing HER3 with an antigen binding molecule according to any one of paragraphs 1 to 27 and detecting the formation of a complex of the antigen binding molecule and HER 3.

44. A method of selecting a subject or stratifying a subject for HER3 targeted drug therapy, the method comprising contacting a sample of the subject in vitro with the antigen binding molecule of any one of paragraphs 1 to 27 and detecting the formation of a complex of the antigen binding molecule with HER 3.

45. Use of an antigen binding molecule as defined in any one of paragraphs 1 to 27 as a diagnostic or prognostic agent in vitro or in vivo.

46. Use of an antigen binding molecule of any one of paragraphs 1 to 27 in a method for detecting, localizing or imaging a cancer, optionally wherein the cancer is selected from: cancers comprising cells that express an EGFR family member, cancers comprising cells that express a solid tumor of HER3, breast cancer, malignant epithelial carcinoma of the breast, ductal carcinoma, gastric carcinoma, malignant epithelial carcinoma of the stomach, gastric adenocarcinoma, colorectal cancer, malignant epithelial carcinoma of the colorectal, colorectal adenocarcinomas, head and neck cancer, squamous cell cancer of the head and neck (SCCHN), lung cancer, malignant epithelial cancer of the lung, squamous cell lung malignant epithelial cancer, ovarian cancer, malignant epithelial cancer of the ovary, serous adenocarcinoma of the ovary, kidney cancer, malignant epithelial cancer of the kidney, clear cell cancer of the kidney, adenocarcinoma of the kidney, papillary cell cancer of the kidney, pancreatic cancer, adenocarcinoma of the pancreas, ductal adenocarcinoma, cervical cancer, squamous cell carcinoma of the cervix, skin cancer, melanoma, esophageal cancer, esophageal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, uterine cancer, endometrial carcinoma, thyroid cancer, malignant epithelial cancer of the thyroid, chromocytoma, cancer of the like, breast cancer, pancreatic adenocarcinoma, breast cancer, colorectal carcinoma, breast cancer, carcinoma of the like carcinoma, carcinoma of the head carcinoma, carcinoma of the like head of the like, head carcinoma, head of the head carcinoma, head of the head, head of the head, head of the head, head of the head, head of the head, head of the head, head of the head, head of the head of, Paraganglioma, bladder cancer, bladder urothelial cancer, prostate adenocarcinoma, sarcoma, and thymoma.

47. An optionally isolated antigen binding molecule capable of binding to HER3, wherein the antigen binding molecule comprises:

(i) a heavy chain variable region (VH) comprising the following CDRs:

has the sequence shown in SEQ ID NO: 43 of the amino acid sequence HC-CDR1

HC-CDR2 having amino acid sequence shown in SEQ ID NO. 46

Has the sequence of SEQ ID NO: 51, HC-CDR 3; and

(ii) a light chain variable region (VL) comprising the following CDRs:

LC-CDR1 having amino acid sequence shown in SEQ ID NO. 91

LC-CDR2 having amino acid sequence shown in SEQ ID NO. 94

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 99.

48. The antigen binding molecule according to paragraph 47, wherein the antigen binding molecule comprises:

(i) a heavy chain variable region (VH) comprising the following CDRs:

HC-CDR1 having amino acid sequence shown in SEQ ID NO. 41

HC-CDR2 having amino acid sequence shown in SEQ ID NO. 45

HC-CDR3 having the amino acid sequence set forth in SEQ ID NO. 48; and

(ii) a light chain variable region (VL) comprising the following CDRs:

LC-CDR1 having the amino acid sequence shown in SEQ ID NO. 88

LC-CDR2 having the amino acid sequence shown in SEQ ID NO. 92

LC-CDR3 having the amino acid sequence shown in SEQ ID NO. 95.

49. The antigen binding molecule of

paragraph

47 or 48, wherein the antigen binding molecule comprises:

50. An optionally isolated antigen binding molecule comprising (i) an antigen binding molecule according to any one of paragraphs 47 to 49, and (ii) an antigen binding molecule capable of binding to an antigen other than HER 3.

51. A Chimeric Antigen Receptor (CAR) comprising the antigen binding molecule of any one of paragraphs 47 to 50.

52. One or more optionally isolated nucleic acids encoding an antigen binding molecule according to any of paragraphs 47 to 50 or a CAR according to paragraph 51.

53. An expression vector or vectors comprising a nucleic acid or nucleic acids according to paragraph 52.

54. A cell comprising an antigen binding molecule according to any one of paragraphs 47 to 50, a CAR according to paragraph 51, a nucleic acid or nucleic acids according to paragraph 52, or an expression vector or expression vectors according to paragraph 29 to paragraph 53.

55. A method comprising culturing a cell comprising a nucleic acid or nucleic acids according to paragraph 52, or an expression vector or expression vectors according to paragraph 53, under conditions suitable for expression of an antigen binding molecule or CAR from the nucleic acid or nucleic acids or expression vector or vectors.

56. A composition comprising an antigen binding molecule according to any one of paragraphs 47 to 50, a CAR according to paragraph 51, a nucleic acid or nucleic acids according to paragraph 52, an expression vector or vectors according to paragraph 53, or a cell of paragraph 54.

57. An antigen binding molecule according to any one of paragraphs 47 to 50, a CAR according to paragraph 51, one or more nucleic acids according to paragraph 52, an expression vector or vectors according to paragraph 53, a cell according to paragraph 54, or a composition according to paragraph 56, for use in a medical or prophylactic method.

58. An antigen binding molecule according to any one of paragraphs 47 to 50, a CAR according to paragraph 51, one or more nucleic acids according to paragraph 52, an expression vector or vectors according to paragraph 53, a cell according to paragraph 54 or a composition according to paragraph 56 for use in a method of treating or preventing cancer.

59. Use of an antigen binding molecule of any one of paragraphs 47 to 50, a CAR of paragraph 51, one or more nucleic acids of paragraph 52, an expression vector or expression vectors of paragraph 53, a cell according to paragraph 54 or a composition according to paragraph 56, in the manufacture of a medicament for use in a method of treating or preventing cancer.

60. A method of treating or preventing cancer, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen binding molecule according to any one of paragraphs 47 to 50, a CAR according to paragraph 51, a nucleic acid or nucleic acids according to paragraph 52, an expression vector or vectors according to paragraph 53, a cell of paragraph 54 or a composition of paragraph 56.

61. The antigen binding molecule, CAR, one or more nucleic acids, expression vector or vectors, cell or composition for use according to paragraph 57 or 58, the use according to paragraph 59 or the method according to paragraph 60, wherein the method further comprises administering an inhibitor of signal transduction mediated by an EGFR family member, optionally wherein the inhibitor of signal transduction mediated by an EGFR family member is an inhibitor of signal transduction mediated by HER2 and/or EGFR.

62. The use, use or method of any of paragraphs 57 to 61 of an antigen binding molecule, CAR, nucleic acid or nucleic acids, expression vector or expression vectors, cell or composition, wherein the cancer is selected from: cancers comprising cells that express an EGFR family member, cancers comprising cells that express HER3, solid tumors, breast cancer, malignant epithelial carcinoma of the breast, ductal carcinoma, gastric cancer, malignant epithelial carcinoma of the stomach, gastric adenocarcinoma, colorectal cancer, malignant epithelial carcinoma of the colorectal, colorectal adenocarcinomas, head and neck cancer, squamous cell cancer of the head and neck (SCCHN), lung cancer, malignant epithelial cancer of the lung, squamous cell lung malignant epithelial cancer, ovarian cancer, malignant epithelial cancer of the ovary, ovarian serous adenocarcinoma, kidney cancer, malignant epithelial cancer of the kidney, clear cell cancer of the kidney, adenocarcinoma of the kidney, papillary cell cancer of the kidney, pancreatic cancer, adenocarcinoma of the pancreas, ductal adenocarcinoma, cervical cancer, squamous cell carcinoma of the cervix, skin cancer, melanoma, esophageal cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, uterine cancer, endometrial cancer, thyroid cancer, malignant epithelial cancer of the thyroid, chromocytoma, pheochromocytoma, breast cancer, pancreatic carcinoma, bile duct carcinoma, pancreatic carcinoma, breast carcinoma, pancreatic carcinoma, breast carcinoma, pancreatic carcinoma, bladder carcinoma, pancreatic carcinoma, bladder carcinoma, pancreatic carcinoma, breast carcinoma, pancreatic carcinoma, bladder carcinoma, pancreatic carcinoma, bladder carcinoma, breast carcinoma, bladder carcinoma, pancreatic carcinoma, bladder, Paraganglioma, bladder cancer, bladder urothelial cancer, prostate adenocarcinoma, sarcoma, and thymoma.

63. A method of inhibiting HER 3-mediated signaling comprising contacting a cell expressing HER3 with an antigen binding molecule according to any one of paragraphs 47 to 50.

64. A method of reducing the number or activity of a cell expressing HER3, the method comprising contacting a cell expressing HER3 with an antigen binding molecule according to any one of paragraphs 47-50.

65. An optionally isolated in vitro complex comprising an antigen binding molecule according to any one of paragraphs 47 to 50 that binds to HER 3.

66. A method comprising contacting a sample containing or suspected of containing HER3 with an antigen binding molecule according to any one of paragraphs 47 to 50 and detecting the formation of a complex of the antigen binding molecule and HER 3.

67. A method of selecting a subject or stratifying a subject for HER3 targeted drug therapy, the method comprising contacting a sample of the subject in vitro with the antigen binding molecule of any one of paragraphs 47 to 50 and detecting the formation of a complex of the antigen binding molecule with HER 3.

68. Use of the antigen binding molecule of any one of paragraphs 47 to 50 as a diagnostic or prognostic agent in vitro or in vivo.

69. Use of an antigen binding molecule of any one of paragraphs 47 to 50 in a method for detecting, localizing or imaging a cancer, optionally wherein the cancer is selected from: cancer comprising cells that express an EGFR family member, cancer comprising cells that expresses HER3, solid tumors of breast, malignant epithelial carcinoma of breast, ductal carcinoma, gastric carcinoma, malignant epithelial carcinoma of stomach, gastric adenocarcinoma, colorectal carcinoma, malignant epithelial carcinoma of colorectal, colorectal adenocarcinoma, head and neck cancer, Squamous Cell Carcinoma of Head and Neck (SCCHN), lung cancer, malignant epithelial carcinoma of lung, squamous cell lung malignant epithelial carcinoma, ovarian cancer, malignant epithelial carcinoma of ovary, serous adenocarcinoma of ovary, kidney cancer, malignant epithelial carcinoma of kidney cell, clear cell carcinoma of kidney cell, adenocarcinoma of kidney cell, papillary cell carcinoma of kidney, pancreatic cancer, adenocarcinoma of pancreatic duct, cervical cancer, squamous cell carcinoma of cervix, skin cancer, melanoma, esophageal cancer, esophageal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, uterine cancer, endometrial carcinoma, thyroid carcinoma, malignant epithelial carcinoma of thyroid, chromocytoma, pheochromocytoma carcinoma, cancer of colon carcinoma of colon, colon carcinoma of pancreas, colon carcinoma of pancreas, bladder, carcinoma of pancreas, carcinoma of the head and colon carcinoma of the like, Paraganglioma, bladder cancer, bladder urothelial cancer, prostate adenocarcinoma, sarcoma and thymoma.

***

The invention includes the combination of aspects and preferred features described unless such combination is clearly not allowed or specifically avoided.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. .

Aspects and embodiments of the present invention will now be illustrated by way of example with reference to the accompanying drawings. Other aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment.

Where nucleic acid sequences are disclosed herein, their reverse complementarity is also expressly contemplated.

The methods described herein may preferably be performed in vitro. The term "in vitro" is intended to cover procedures performed with cultured cells, while the term "in vivo" is intended to cover procedures used/on intact multicellular organisms.

Brief Description of Drawings

Embodiments and experiments illustrating the principles of the present invention will now be discussed with reference to the accompanying drawings.

Fig. 1A and 1b. histograms show staining of cells by anti-HER 3 antibody as determined by flow cytometry. The histograms show the staining of HEK293 cells (not expressing HER3), or HEK293 cells overexpressing HER3 (HEK293 HER 3O/E) by the (1A, 1B) anti-HER 3 antibody clone 10D1 and the (1B) anti-HER 3 antibody clone LJM 716.

Fig. 2A and 2b. histograms show staining of cells by anti-HER 3 antibody as determined by flow cytometry. The histograms show the staining of HEK293 cells (not expressing HER3) by the (2A, 2B) anti-HER 3 antibody clone 4-35-B2 and (2B) anti-HER 3 antibody clone LJM716, or HEK293 cells overexpressing HER3 (HEK293 HER 3O/E).

Fig. 3A and 3b. histograms show staining of cells by anti-HER 3 antibody as determined by flow cytometry. Histograms show staining of HEK293 cells (not expressing HER3), or HEK293 cells overexpressing HER3 (HEK293 HER 3O/E), with anti-HER 3 antibody clone 4-35-B4(3A, 3B) and anti-HER 3 antibody clone LJM716 (3B).

Figure 4 histogram shows staining of cells by anti-HER 3 antibody as determined by flow cytometry. The histograms show staining of HEK293 cells (not expressing HER3) or HER3 over-expressed HEK293 cells (HEK293 HER 3O/E) by the anti-HER 3 antibody clone 10a 6.

FIGS. 5A and 5B show the anti-HER 3 antibody clone 10D1 in combination with (5A) human, mouse, rat and cynomolgus monkey HER3 and (5B) human EGFResults of ELISA analysis of R and human HER2 binding. EC (EC) ₅₀ The values are shown in the figure.

Figures 6A and 6B as a graph show the results of an ELISA analysis of the anti-HER 3 antibody clone 4-35-B2 binding to (6A) human, mouse, rat and cynomolgus monkey HER3 and (6B) human EGFR and human HER 2.

Fig. 7A and 7B as a graph showing the results of ELISA analysis of anti-HER 3 antibody clone 4-35-B4 binding to (7A) human HER3, human EGFR and human HER2 and (7B) human, mouse, rat and cynomolgus monkey HER 3.

Figure 8. representative sensorgram shows the results of affinity analysis of anti-HER 3 antibody clone 10D1 binding to human HER 3. Shows Kon, Koff and K _D 。

Figure 9. representative sensorgram shows the results of affinity analysis of the binding of anti-HER 3 antibody clone 4-35-B2 to human HER 3.

Figure 10. representative sensorgram shows the analysis of the binding affinity of anti-HER 3 antibody clone 4-35-B4 to human HER 3.

Figure 11 shows the results of differential scanning fluorescence analysis of the stability of anti-HER 3 antibody clone 10D 1.

Figure 12 shows the results of analysis of recombinantly expressed anti-HER 3 antibody clone 10D1 by size exclusion chromatography.

Figure 13 shows the results of expression of anti-HER 3 antibody clone 10D1 as analyzed by SDS-PAGE and western blot. Lanes: m1 ═ TaKaRa protein designation, cat No. 3452; m2 ═ GenScript protein marker, cat No. M00521; 1 ═ reducing conditions; 2 ═ non-reducing conditions; p ═ positive control: human IgG1, kappa (Sigma cat # I5154). For Western blotting, goat anti-human IgG-HRP (GenScript cat # A00166) and goat anti-human kappa-HRP (SouthhnBiotech cat # 2060-05) were used as primary antibodies.

Fig. 14A and 14B. Representative sensorgrams and tables show competition results for competition binding with HER3 between different anti-HER 3 antibody clones.

Figure 15 shows the results of an analysis of the inhibition of the interaction between HER3 and HER2 by anti-HER 3 antibody clone 10D1 as determined by ELISA.

Fig. 16A and 16b. tables and histograms show gene and protein expression of EGFR protein family members and their ligands for different cancer cell lines.

Figure 17 image shows effect of anti-HER 3 antibody clone 10D1 treatment on HER3 mediated signaling in N87 and FaDu cells phosphorylating western blot analysis results. Un ═ untreated; t-treated with anti-HER 3 antibody clone 10D 1.

Figure 18. images and graphs show the results of an analysis of the effect of anti-HER 3 antibody clone 10D1 treatment on HER 3-mediated signaling in FaDu cells using phosphoprotein antibody array assay kit. Untreated versus untreated FaDu cells; FaDu cells treated with anti-HER 3 antibody clone 10D 1.

Figures 19A and 19b images show the percentage of cell fusion of the indicated cell lines relative to untreated control conditions (100%) determined by CCK8 analysis after incubation in the presence of anti-HER 3 antibody clone 10D 1. (19A) Shows the results obtained from N87 cells, while (19B) shows the results obtained from FaDu cells.

Figure 20 shows the results of tumor volume analysis over time in a mouse gastric cancer model derived from the N87 cell line. anti-HER 3 antibody clone 10D1 was intraperitoneally administered once every two weeks at a dose of 500 μ g for a total of 10 doses. Control treated groups received an equal volume of PBS (vehicle).

FIG. 21 shows the results of tumor volume analysis over time in a mouse gastric cancer model derived from the N87 cell line. anti-HER 3 antibody clone 4-35-B2 was intraperitoneally administered at a dose of 11mg/kg once a week for a total of 4 doses. Control treated groups received an equal amount of isotype control antibody (isotype).

Figure 22 shows the results of tumor volume analysis over time in a mouse gastric cancer model derived from the SNU16 cell line. anti-HER 3 antibody clone 9D1 was intraperitoneally administered once every two weeks at a dose of 500 μ g for a total of 10 doses. Control treated groups received an equal volume of PBS (vehicle).

Figure 23 shows the results of tumor volume analysis over time in a FaDu cell line-derived head and neck squamous cell carcinoma mouse model. anti-HER 3 antibody clone 10D1 was injected intraperitoneally once weekly at 500 μ g doses for a total of 4 doses. Control treated groups received an equal volume of PBS (vehicle) or the same dose of isotype control antibody (isotype).

Figure 24 shows the results of tumor volume analysis over time in a FaDu cell line-derived head and neck squamous cell carcinoma mouse model. anti-HER 3 antibody clone 10D1 was intraperitoneally administered at a dose of 500 μ g every two weeks for a total of 8 doses. Control treated groups received an equal volume of PBS (vehicle).

Figure 25 shows the results of tumor volume analysis over time in a mouse ovarian cancer model derived from the OvCAR8 cell line. anti-HER 3 antibody clone 9D1 was intraperitoneally administered once every two weeks at a dose of 500 μ g for a total of 10 doses. Control treated groups received an equal volume of PBS (vehicle).

FIG. 26 shows the results of tumor volume analysis over time of HCC-95 cell line derived from a mouse squamous cell carcinoma model. anti-HER 3 antibody clone 4D1 was intraperitoneally administered at a dose of 500 μ g every two weeks for a total of 10 doses. Control treated groups received an equal volume of PBS (vehicle).

Figure 27 shows the results of tumor volume analysis over time in a mouse lung adenocarcinoma model derived from the a549 cell line. anti-HER 3 antibody clone 10D1 was intraperitoneally administered once every two weeks at a dose of 500 μ g for a total of 10 doses. Control treated groups received an equal volume of PBS (vehicle).

Figure 28 shows the results of a tumor volume analysis over time in mouse lung adenocarcinomas derived from the a549 cell line. anti-HER 3 antibody clone 4-35-B2 was intraperitoneally injected once every two weeks at a dose of 500. mu.g for a total of 10 doses. Control treated groups received an equal volume of PBS (vehicle).

Figure 29 shows the results of tumor volume analysis over time in a mouse renal cell carcinoma model derived from the ACHN cell line. anti-HER 3 antibody clone 7D1 was intraperitoneally injected once every two weeks at a dose of 500 μ g for a total of 10 doses. Control treated groups received an equal volume of PBS (vehicle).

Figure 30 histogram shows staining of cells by anti-HER 3 antibody clone 10D1_ c89 as determined by flow cytometry. The histograms show staining of HEK293 cells (not expressing HER3) or HEK293 HER3 overexpressed cells (HEK293 HER 3O/E).

Figure 31 histogram shows staining of cells by anti-HER 3 antibody clone 10D1_ c90 as determined by flow cytometry. The histograms show staining of HEK293 cells (not expressing HER3) or HEK293 HER3 over-expressed cells (HEK293 HER 3O/E).

Figure 32 histogram shows staining of cells by anti-HER 3 antibody clone 10D1_ c91 as determined by flow cytometry. The histograms show staining of HEK293 cells (not expressing HER3) or HEK293 HER3 over-expressed cells (HEK293 HER 3O/E).

Figures 33A and 33b shows the results of ELISA analysis of the binding of the anti-HER 3 antibody 10D1 variant clone to human HER 3. (33A) Binding of anti-HER 3 antibody clones 10D1 (referred to as 10D1P), 10D1_ c75, 10D1_ c76, 10D1_ c77, 10D1_ c78, 10D1_11B (referred to as v11B78L), 10D1_ c85, 10D1_ c85o1, 10D1_ c85o2, 10D1_ c87, 10D1_ c89, 10D1_ c90, 10D1_ c91, 10D1_ c93, LJM716 and hIgG (negative control) was shown. (33B) The data shown is the same as 33A, but only for clones 10D1_ c89, 10D1_ c90, 10D1_ c91, and LJM 716.

Figures 34A and 34b show the results of analysis of recombinantly expressed anti-HER 3 antibody 10D1 variant clones by size exclusion chromatography. (34A) Results of anti-HER 3 antibody clones 10D1_ C93, 10D1_ C75, 10D1_ C76, 10D1_ C77, 10D1_ C78, 10D1_11B (referred to as C78 v11B), 10D1_ C85, 10D1_ C85o1, 10D1_ C85o2, 10D1_ C89, 10D1_ C90, 10D1_ C91 and 10D1_ C93 are shown. (34B) The data shown is the same as 33A, but only for clones 10D1_ c89, 10D1_ c90, 10D1_ c 91.

Figures 35A to 35c show the results of differential scanning fluorescence analysis of the stability of the anti-HER 3 antibody 10D1 variant clone. (35A) Results for anti-HER 3 antibody clones LJM716 (also known as eggeminmumab), 10D1 (known as 10D1 (parental)), 10D1_ c75, 10D1_ c76, 10D1_ c77, and 10D1_ c78 are shown. (35B) Results are shown for 10D1_ c85o2, 10D1_ c87, 10D1_ c89, 10D1_11B (referred to as c78_ V11B), 10D1_ c85 and 10D1_ c85o 1. (35C) Results for 10D1_ c90, 10D1_ c91, and 10D1_ c93 are shown.

Fig. 36A to 36m representative sensorgrams show the results of affinity analysis of the anti-HER 3 antibody 10D1 variant clone to human HER 3. Shows Kon, Koff and K _D . (36A) Shows the results for clone 10D1_ c89, (36B) shows the results for clone 10D1_ c90,(36C) results for clone 10D1_ c91 were shown, (36D) results for clone 10D1_ c11B were shown, (36E) results for clone 10D1_ c85o2 were shown, (36F) results for clone 10D1_ c87 were shown, (36G) results for clone 10D1_ c93 were shown, (36H) results for clone 10D1_ c76 were shown, (36I) results for clone 10D1_ c77 were shown, (36J) results for clone 10D1_ c78 were shown, (36K) results for clone 10D1_ c75 were shown, (36L) results for clone 10D1_ c85 were shown, and (36M) results for clone 10D1_ c85o1 were shown.

Figure 37 table summarizes the characteristics of the anti-HER 3 antibody 10D1 variant clone with respect to safety and developability.

Fig. 38A and 38B Fc modified anti-HER 3 antibody clone 10D1 containing GASDALIE and LCKC substitutions in the CH2 region, its biolayer interferometry (38A) and thermal stability (38B) analyses. (38A) BLI shows a representative sensorgram showing the analysis of affinity of binding to Fc γ RIIIa by Fc modified anti-HER 3 antibody clone 10D 1. Shows Kon, Koff and K _D 。

Figures 39A and 39B representative sensorgrams show the results of affinity analysis of binding to Fc γ RIIIa by (39A) non-Fc modified anti-HER 3 antibody clone 10D1 and (39B) Fc modified anti-HER 3 antibody clone 10D1 containing a GASD substitution in the CH2 region. Shows Kon, Koff and K _D 。

Figure 40 shows the results of differential scanning fluorescence analysis of the stability of anti-HER 3 antibody clone 10D1GASD variant.

Figures 41A and 41b. tables show the binding affinity of anti-HER 3 antibody clones 10d1f. fca and 10d1f. fcb (GASDALIE-LCKC variant) for mouse and human Fc receptors compared to silent variant N297Q, isotype variant and commercial antibody. ND-undetermined K due to Low binding affinity _D 。

Fig. 42A and 42b histograms show staining of cells by anti-HER 3 antibody as determined by flow cytometry. Histograms show staining of HEK293 cells (not expressing HER3) or HEK293 HER3 overexpressing cells (HEK293 HER 3O/E) by (42A) anti-HER 3 antibody clone 10D1f. fca and (42B) anti-HER 3 antibody clone 10D1 and LJM-716.

FIG. 43 shows anti-HER 3 antibody clone 10D1F. FcA in combination with human EGFR (HER1) and human HEResults of ELISA analysis of R2 binding. Show EC ₅₀ The value is obtained.

Figure 44 histogram shows staining of cells by anti-HER 3 and anti-HER 4 antibodies as determined by flow cytometry. Histograms show staining of HEK293 HER4 overexpressing cells with anti-HER 3 antibody clone 10d1f. fca, anti-HER 3 antibodies LJM-716 and MM-121, and a commercial anti-HER 4 antibody.

Figure 45 this figure shows the results of an ELISA assay of anti-HER 3 antibody clone 10d1f. fca binding to human, mouse, rat and cynomolgus monkey HER 3. Show EC ₅₀ The value is obtained.

Representative sensorgrams show the results of affinity analysis of anti-HER 3 antibody clones (46A)10d1f. fca and (46B)10d1f. fcb binding to human HER 3. Shows Kon, Koff and K _D 。

Fig. 47A and 47B show the results of analyzing the stability of anti-HER 3 antibody clones (47A)10d1f. fca and (47B)10d1f. fcb by differential scanning fluorescence analysis.

Fig. 48A and 48B show the results of clonal purity of anti-HER 3 antibodies (48A)10d1f. fca and (48B)10d1f. fcb as analyzed by size exclusion chromatography.

Figures 49A and 49b representative sensorgrams and tables show results of HER3 binding competition assays between anti-HER 3 antibody clone 10d1f. fca and anti-HER 3 antibodies M-05-74 and M-08-11.

Figure 50 figure and table show the results of pharmacokinetic analysis of anti-HER 3 antibody clone 10D1 in mice.

Figures 51A to 51F-graphs show the effect of anti-HER 3 antibody clone 10D1 treatment on mouse blood cell counts (51A), electrolyte index (51B), and hepatotoxicity, nephrotoxicity, and pancreatic toxicity indices (51C-51F). The left bar represents vehicle control and the right bar represents 10D1 treatment. The dotted line indicates the end of the reference range of Charles River. Indicators of hepatotoxicity, nephrotoxicity, and pancreatic toxicity include alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), Blood Urea Nitrogen (BUN), Creatinine (CREA), alkaline phosphatase (ALP), Glucose (GLU), Calcium (CAL), total Bilirubin (BIL), Total Protein (TPR), and Albumin (ALB).

Figure 52. images and tables show the results of analysis of the anti-HER 3 antibody clone 10d1f. fca and antibodies MM-121, LJM-716 and Roche (Roche) M05 inhibiting the interaction between HER2 and HER3 as determined by ELISA.

Figure 53. images and tables show the results of analysis of the anti-HER 3 antibody clone 10d1f. fca and antibodies MM-121 and LJM716 inhibiting the interaction between EGFR and HER3 as determined by ELISA.

Figure 54 graphs and tables show the results of analysis of anti-HER 3 antibody clones 10d1f.fca (10d1f.a), 10d1f.fcb (10d1f.b), 10D1F-hIgG1(N297Q), and anti-HER 3 antibodies LJM-716 and selitumab (MM-121) to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Show EC ₅₀ The value is obtained.

Fig. 55A to 55C images show the results of HER3 mediated signaling by phosphowestern blot analysis of anti-HER 3 antibody treatment in (55A) N87, (55B) FaDu and (55C) OvCar8 cells.

Fig. 56A and 56B. images and tables show the results of pharmacokinetic analysis of anti-HER 3 antibody clones (56A)10d1f. fca and (56B)10d1f. fcb in mice. Parameters are as follows: maximum concentration (C) _max )，T _max AUC (0-336 hr), AUC (0-infinity), half-life (t) _1/2 ) Clearance (CL), distribution volume (V) at steady state _ss )。

Figures 57A to 57D. images and tables show the results of pharmacokinetic analysis of anti-HER 3 antibody clones 10d1f. fca and 10d1f. fcb in rats at (57A)10mg/kg, (57B)25mg/kg, (57C)100mg/kg and (57D)250 mg/kg. Parameters are as follows: maximum concentration (C) _max )，T _max AUC (0-336 hr), AUC (0-infinity), half-life (t) _1/2 ) Clearance (CL), distribution volume in steady state (V) _ss )。

Fig. 58A-58F, as shown for anti-HER 3 antibody clone 10D1F. fca or 10D1F. fcb at 200ug (-10 mg/kg), 500ug (-25 mg/kg), 2mg (-100 mg/kg), or 5mg (-250 mg/kg) of therapeutic effect on (58A, 58B) red blood cell index, (58C) white blood cell index, (58D) hepatotoxicity, (58E) kidney and pancreas index, and (58F) electrolyte index.

Fig. 59A to 59D as a graph shows the effect of treatment with anti-HER 3 antibody clone 10D1f. fca on the percentage of tumor inhibition in vitro mouse cancer models of N87 cells (gastric cancer), HCC95 cells (lung cancer), FaDu cells (head and neck cancer), SNU-16 cells (gastric cancer), a549 cells (lung cancer), OvCar8 cells (ovarian cancer), ACHN cells (kidney cancer), and HT29 cells (colorectal cancer) compared to (59A and 59B) anti-HER 3 antibody serlizumab and ltjm-716 and (59C and 59D) EGFR family therapy cetuximab, trastuzumab, and pertuzumab.

Figure 60 shows the results of tumor volume analysis over time following treatment with the indicated concentrations of antibody once every two weeks for six weeks in a549 cell line derived lung adenocarcinoma mouse model (n-6, vehicle control n-8). Triangles along the x-axis indicate antibody administration.

Figure 61 shows the results of analysis of tumor volume over time following six weeks of weekly treatment with the indicated concentrations of antibody in a FaDu cell line derived head and neck squamous cell carcinoma mouse model (n-6). Triangles along the x-axis indicate antibody administration.

Figure 62 shows the results of analysis of tumor volume over time following six weeks of weekly treatment with the indicated concentrations of antibody in an OvCAR8 cell line derived ovarian cancer mouse model (n-6). Triangles along the x-axis indicate antibody administration.

FIGS. 63A to 63D. boxplots show the results of the activation of the genome enrichment assay pathway in an in vitro phosphorylation assay of 10D1F. FcA, LJM-716 or Selizumab-treated cancer cell lines. 63A shows the results obtained from N87 cells, 63B shows the results obtained from A549 cells, 63C shows the results obtained from OvCar8 cells, and 63D shows the results obtained from FaDu cells.

Figure 64 image shows the results of anti-HER 3 antibody treatment on HER3 mediated signaling in a549 cells by anti-phosphorylation western blot analysis at the indicated time points.

Figure 65 shows that 10d1f. fca or pertuzumab inhibits HER2 as determined by the PathHunter pertuzumab bioassay: results of HER3 interaction. IC50(M) values are shown.

Fig. 66A to 66C histograms show the results of analysis of EGFR, HER2, and HER3 expression by (66A) BCPAP (66B) BHT101 and (66C) SW1736 cells. 1 ═ unstained cells, 2 ═ isotype control, 3 ═ cetuximab, 4 ═ trastuzumab, 5 ═ 10d1f. fca.

FIGS. 67A to 67C. images show analysis of different anti-ErbB antibodies for in vitro inhibition of BRAF ^V600E Results of the ability of the mutant thyroid cancer cell lines to proliferate. 67A shows the results obtained from BHT101 cells, 687 shows the results obtained from BCPAP cells, and 67C shows the results obtained from SW1736 cells.

Fig. 68A to 68c. images show that 10d1f. fca alone or in combination with vemurafenib (vemurafenib) inhibits BRAF in vitro ^V600E And (3) analysis results of the proliferation capacity of the mutant thyroid cancer cell line. 68A shows the results obtained from BHT101 cells, 68B shows the results obtained from SW1736 cells, and 68C shows the results obtained from BCPAP cells.

FIGS. 69A to 69℃ tables show representative hematological characteristics of BALB/c mice following administration of 10mg/kg, 25mg/kg, 100mg/kg, or 250mg/kg of 10D1F. FcA or an equal volume of PBS. 69A shows the results of analyzing red blood cells, 69B shows the results of analyzing white blood cells, and 69C shows the results associated with analyzing liver, kidney, pancreas function and electrolyte levels. RBC ═ red blood cells, MVC ═ mean red blood cell volume, MCH ═ mean red blood cell hemoglobin, MCHC ═ mean particulate hemoglobin concentration, WBC ═ white blood cells, ALB ═ albumin, ALT ═ alanine aminotransferase, ALP ═ alkaline phosphatase, CREA ═ creatinine, BUN ═ blood urea nitrogen, GLU ═ glucagon, AMY ═ amylase, NA ═ sodium, K ═ potassium, P ═ phosphorus, CA ═ calcium.

Fig. 70A to 70c. tables show representative hematology characteristics of SD rats at designated time points after administration of 250mg/kg 10d1f. fca or an equal volume of PBS. 70A shows the results of analyzing red blood cells, 70B shows the results of analyzing white blood cells, and 70C shows the associated results of analyzing liver, kidney, pancreas function and electrolyte levels. RBC ═ red blood cell, MVC ═ mean red blood cell volume, MCH ═ mean red blood cell hemoglobin, MCHC ═ mean corpuscular hemoglobin concentration, WBC ═ white blood cells, ALB ═ albumin, ALP ═ alkaline phosphatase, CREA ═ creatinine, BUN ═ blood urea nitrogen, GLU ═ glucagon, AMY ═ amylase, NA ═ sodium, K ═ potassium, P ═ phosphorus, CA ═ calcium.

Figure 71 image shows the results of analysis of the effect of 10d1f. fca treatment on HER3 mediated in vivo signaling in FaDu or OvCar8 cell derived tumor cells by phosphowestern blot assay.

Figure 72 box plot shows the results of an analysis of internalization of different anti-ErbB antibodies by a given cell line.

Fig. 73A and 73b histograms and tables show the results of analysis of internalization of 10d1f. fca or trastuzumab by the indicated cell lines at different time points as determined by flow cytometry. 73B shows the median fluorescence intensity and the percentage of PE positive cells determined from the histogram shown in 73A.

Figure 74 shows tumor volume analysis over time in a N87 cell line derived gastric cancer mouse model following biweekly treatment with indicated concentrations of the indicated anti-ErbB antibody for six weeks (N-6). Triangles along the x-axis indicate antibody administration.

Fig. 75A and 75b pictures show immunohistochemical staining of malignant and normal human tissues using 10d1f. 75A and 75B show staining of different tissues.

Figure 76 image shows immunohistochemical staining of a549 tumor xenograft sections with 10D1F or rabbit multi-anti-HER 3 antibody at specified magnifications. Only minor control staining is shown.

Figures 77A and 77b, bar graphs show the results of an assay showing the ability of anti-ErbB antibodies to inhibit the proliferation of the indicated cancer cell lines in vitro, when antibody serum concentrations reach Cmax after intraperitoneal injection into mice at 25 mg/kg. 77A and 77B show the results obtained using different cell lines.

Figures 78A to 78F representative sensor plots show the results of affinity analysis of the anti-HER 3 antibodies 10d1f.a (78A,78B), MM-121(78C,78D) and LJM-716(78E,78F), binding to human HER3 in the absence of human NRG1 (78A,78C,78E), and in the presence of human NRG1 (78B,78D, 78F). Shows Kon, Koff and K _D 。

Figure 79 image shows the analysis of tumor volume over time in a patient-derived ovarian cancer xenograft model comprising CLU-NRG1 fusions following biweekly treatments with 25mg/kg of 10D1F or an isotype-matched control antibody (n-10 per treatment group). Triangles along the x-axis indicate antibody administration.

Examples

In the following examples, the inventors describe the generation of novel anti-HER 3 antibody clones targeting specific regions of interest in the HER3 molecule, as well as the biophysical and functional characteristics of these antigen binding molecules and the evaluation of therapeutics.

Example 1: HER3 target design and production of anti-HER 3 antibody hybridomas

The inventors selected two regions in the extracellular region of human HER3 (SEQ ID NO: 9) for the production of monoclonal antibodies that bind HER 3.

1.1 Generation of hybridomas

Female BALB/c mice of approximately 6 weeks of age were obtained from InVivos (Singapore). Animals were housed in specific pathogen-free conditions and cultured according to Institutional Animal Care and Use Committee (IACUC) guidelines.

To generate hybridomas, mice are immunized with antigenic peptides, recombinant target proteins, or a proprietary mixture of cells expressing the target proteins.

Mice were boosted with antigen cocktail for three consecutive days or only one day prior to harvesting the spleen for fusion. 24 hours after the last booster immunization, total splenocytes were isolated using the clonacyl-HY hybridoma cloning kit according to the manufacturer's instructions (stem cell technology, canada) and fused with PEG to the myeloma cell line p3x63.ag8.653(ATCC, usa).

In ClonaCell-HY Medium C (Stem cell technology, Canada), 5% CO ₂ The fused cells were cultured overnight at 37 ℃ in an incubator. The next day, the fused cells were centrifuged and resuspended in 10ml of ClonaCell-HY medium C, and then gently mixed with 90ml of semisolid methylcellulose-based ClonaCell-HY medium D (Stem cell technology, Canada) containing HAT component to combine hybridoma selection and cloning into one step.

The fused cells were then seeded into 96-well plates at 5% CO ₂ Growth was carried out in an incubator at 37 ℃. After 7-10 days, individual hybridoma clones were isolated and the supernatants were screened by enzyme-linked immunosorbent assay (ELISA) and fluorescence activated cell sorting (FAC) to select antibody-producing hybridomas.

1.2 antibody variable region amplification and sequencing

Total RNA was extracted from hybridoma cells using TRIzol reagent (Life technologies, Inc., USA) according to the manufacturer's experimental procedures. According to the manufacturer's instructions, the SMARTer RACE 5'/3 ' kit (Clontech) was used ^TM U.S. Pat. No.; USA) double-stranded cDNA was synthesized. Briefly, 1. mu.g of total RNA was used to generate full-length cDNAs using 5' -RACE CDS primers (provided in the kit), and 5' adapters (SMARTer II A primers) were then incorporated into each cDNA according to the manufacturer's instructions. The cDNA synthesis reaction includes: 5 XFirst Strand buffer, DTT (20mM), dNTP Mix (10mM), RNase inhibitor (40U/. mu.l) and SMARTScripte reverse transcriptase (100U/. mu.l).

SeqAmp DNA polymerase (Clontech) was used ^TM U.S.A.) to amplify cDNA for race backup. The amplification reaction contained SeqAmp DNA polymerase, 2X Seq AMP buffer, 5' universal primers (complementary to the adaptor sequence) provided in the 5' smart Race kit, and 3' primers annealing to the corresponding heavy or light chain constant region primers. The 5' constant region was designed based on the following previously reported primer mix: krebber et al, J.Immunol.D. 1997; 201:35-55, Wang et al, J Immunol 2000,233; 167-; 350:183-193. The following heating methods were used: circulation 1 min before denaturation at 94 ℃; 35 cycles of 94 ℃, 30s, 55 ℃, 30s and 72 ℃, 45 s; final extension, 72 ℃,3 min.

The resulting approximately 550bp VH and VL PCR products were cloned into pJET 1.2/blunt vector using the CloneJET PCR cloning kit (Sammerfet technology, USA) for transformation of highly competent E.coli DH5 alpha. Plasmid DNA was prepared from the resulting transformants using the Miniprep kit (Qiagene, Germany) and sequenced. DNA sequencing was performed by AITbiotech. These sequencing data were analyzed using the international IMGT (ImMunoGeneTics, immunologetics) information system (LeFranc et al, nucleic acids research (2015)43 (database album): D413-22) to characterize the individual CDR and framework sequences. The 5' -signal peptides of VH and VL were identified by SignalP (v 4.1; Nielsen, eds. Kihara, D.: Protein Function Prediction 59-73, Methods in Molecular Biology 1611, Springer 2017).

Four monoclonal anti-HER 3 antibody clones were selected for further development: 10D1, 10A6, 4-35-B2, and 4-35-B4.

A humanized version of 10D1 was designed in silico by grafting Complementarity Determining Regions (CDRs) into VH and VL comprising human antibody framework regions and further optimizing antigen binding by the yeast display method.

For yeast display, the humanized sequence was converted to single-chain fragment variable (scFv) form by Polymerase Chain Reaction (PCR) and used as a template to generate a library of mutants by random mutagenesis. The mutated PCR library was then electroporated into yeast along with the linearized pCTcon2 vector to generate a yeast library. The library was stained with human HER3 antigen and the best binders were selected. After 4-5 rounds of sorting, individual yeast clones were sequenced and unique antibody sequences were identified.

Example 2: antibody production and purification

2.1 cloning of VH and VL into expression vectors:

the DNA sequences encoding the heavy and light chain variable regions of the anti-HER 3 antibody clone were subcloned into pmAbDZ _ IgG1_ CH and pmAbDZ _ IgG1_ CL (english corporation, usa) eukaryotic expression vectors to construct a human-murine chimeric antibody.

Alternatively, the DNA sequences encoding the heavy and light chain variable regions of the anti-HER 3 antibody clone were subcloned into pFUSE-CHIg-hG1 and pFUSE2ss-CLIg-hk (Engyl corporation, USA) eukaryotic expression vectors to construct human-murine chimeric antibodies. The human IgG1 constant region encoded by pFUSE-CHIg-hG1 included D356E, L358M substitutions (positions numbered according to European Union numbering) in the CH3 region relative to the human IgG1 constant region (IGHG 1; UniProt: P01857-1, v 1; SEQ ID NO: 176). pFUSE2ss-CLIg-hk encodes the kappa constant region of human IgG1 light chain (IGCK; Unit: P01834-1, v 2).

The SeqAmp enzyme (Clontech) was used according to the manufacturer's protocol ^TM U.S. Pat. No. 4) the variable region was amplified from the cloning vector together with the signal peptide. Forward and reverse primers overlapping 15-20bp with the appropriate region in VH or VL were used, plus 6bp at the 5' end as restriction sites. The DNA insert and vector were digested with the manufacturer's recommended restriction enzymes to ensure that no frameshifts were introduced and inserted into the respective plasmids using T4 ligase (siemer fly technologies, usa). The molar ratio of DNA insert to vector was 3: 1 for ligation.

2.2 expression of antibodies in mammalian cells

Antibodies were expressed using either 1) the Expi293 transient expression system kit (Life technologies, USA), or 2) the HEK293-6E transient expression system (CNRC-NRC, Canada) according to the manufacturer's instructions.

1) Expi293 transient expression system:

cell line maintenance:

HEK293F cells (Expi293F) were obtained from life technologies (usa). 8% CO with shaking platform in serum-free, protein-free, chemically-defined medium (Expi293 expression Medium, Sammerfiell, USA) supplemented with 50IU/ml penicillin and 50. mu.g/ml streptomycin (Gicago, USA) at 37 ℃ ₂ And cells were cultured in an 80% humidified incubator.

Transfection:

expi293F cells were transfected with expression plasmids using the expifactamine 293 kit (giucan, usa) according to the manufacturer's protocol. Briefly, 1 day prior to transfection, the maintained cells were medium exchanged by centrifugation of the culture to remove the antibiotic, and the cell pellet was resuspended in fresh medium without antibiotic. On the day of transfection, 2.5X 10 of each transfection was performed ⁶ Viable cells/ml were inoculated in shake flasks. The DNA-Expifeacylamine complex was formed in reduced serum medium Opti-MEM (Gico, USA) at room temperature for 25 min, and then added to the cells. The enhancer was added to the transfected cells 16-18 hours after transfection. An equal amount of medium was added to the transfectants on day 4 post-transfection to prevent cell aggregation. The transfectants were harvested on day 7 by centrifugation at 4000 Xg for 15 minutes and filtered through a 0.22 μm sterile filter.

2) HEK293-6E transient expression system

Cell line maintenance:

HEK293-6E cells were from the Canada national research Committee. Cells were cultured at 37 ℃ in serum-free, protein-free, chemically defined Freestyle F17 medium (Invitrogen, USA) supplemented with 0.1% Kolliphor-P188 and 4mM L-glutamine (Gico, USA) and 25. mu.g/ml G-418 in 5% CO2 and 80% humidified incubator with a shaking platform.

Transfection：

HEK293-6E cells were transfected with expression plasmids using PEIpro (Polyplus, USA) according to the protocol of their manufacturer. Briefly, 1 day prior to transfection, the maintained cells were medium exchanged by centrifugation to remove the antibiotic and the cell pellet was resuspended in fresh medium without antibiotic. On the day of transfection, 1.5-2X 10 of the DNA fragment is required for each transfection ⁶ Cells/ml viable cells were seeded in shake flasks. The DNA and PEIProTM were mixed at a ratio of 1:1, and then a complex was formed in F17 medium at room temperature for 5 minutes, followed by addition to the cells. At 24-48 hours post-transfection, 0.5% (w/v) of trypsin N1 was added to the transfectants. On days 6-7The transfectants were harvested by centrifugation at 4000 Xg for 15 minutes and the supernatant filtered through a 0.22 μm sterile filter.

Cells were transfected with vectors encoding the following combinations of polypeptides:

2.3 antibody purification

Affinity purification, buffer exchange and storage:

the antibody secreted into the culture supernatant by the transfected cells was purified using a liquid chromatography system AKTA Start (GE healthcare Co., UK). Specifically, the supernatant was loaded onto a HiTrap G protein chromatography column (GE healthcare, UK) at an association rate of 5ml/min, and then washed with 10 column volumes of washing buffer (20mM sodium phosphate, pH 7.0). Bound monoclonal antibody was eluted with elution buffer (0.1M glycine, pH 2.7) and the eluate was fractionated into collection tubes containing the appropriate amount of neutralization buffer (1M Tris, pH 9). The neutralization elution buffer containing the purified mAb was exchanged for PBS using a 30K MWCO protein concentrator (seymefel, usa) or a 3.5K MWCO dialysis cassette (seymefel, usa). The monoclonal antibodies were sterilized through a 0.22 μm filter, aliquoted and stored by quick freezing at-80 ℃.

2.4 antibody purity assay

Size Exclusion Chromatography (SEC):

antibody purity was analyzed by Size Exclusion Chromatography (SEC) in PBS running buffer using Superdex 20010/30GL column (GE healthcare, uk) on AKTA Explorer liquid chromatography system (GE healthcare, uk). 150 μ g of antibody dissolved in 500 μ l PBS pH 7.2 was injected into the column at room temperature at a flow rate of 0.75 ml/min. Proteins elute according to molecular weight.

The results of anti-HER 3 antibody clone 10D1 (example 2.2 [1]) are shown in FIG. 12.

The results obtained from the different 10D1 variant clones are shown in fig. 34.

Sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE);

antibody purity was also analyzed by SDS-PAGE under reducing and non-reducing conditions according to standard methods. Briefly, proteins were resolved using a Mini-Protean electrophoresis system (Bio-Rad, USA) using a 4% -20% TGX protein gel (Bio-Rad, USA). For non-reducing conditions, protein samples were denatured by mixing them with 2x Laemmli sample buffer (bole, usa) and boiling for 5-10 minutes at 95 ℃ before loading onto the gel. For reducing conditions, 2x sample buffer containing 5% β -mercaptoethanol (β ME) or 40mM DTT (dithiothreitol) was used. Electrophoresis was performed in SDS electrophoresis buffer (25mM Tris,192mM glycine, 1% SDS, pH 8.3) at a constant voltage of 150V for 1 h.

Immunoblotting

Protein samples (30. mu.g) were fractionated by SDS-PAGE and transferred to nitrocellulose membranes as described above. The membrane was then blocked and immunoblotted with antibody overnight at 4 ℃. After washing 3 times in PBS-Tween, the membrane was then incubated with horseradish peroxidase (HRP) -conjugated secondary antibody for 1 hour at room temperature. The results were visualized by a chemiluminescent Pierce ECL substrate Western blot detection system (american siemer fly science) and autoradiographic film (kodak XAR film).

Primary antibodies used for detection were goat anti-human IgG-HRP (GenScript Cat No. A00166) and goat anti-human kappa-HRP (SouthhnBiotech Cat No. 2060-05).

The results of anti-HER 3 antibody clone 10D1 (example 2.2 [1]) are shown in FIG. 13. 10D1 was readily expressed, purified and processed at high concentrations.

Example 3: biophysical characterization

3.1 analysis of cell surface antigen binding by flow cytometry

Wild-type HEK293 cells (which do not express high levels of HER3) and HEK293 cells transfected with a vector encoding human HER3 (i.e. HEK293 HER O/E cells) were incubated with 20 μ g/ml anti-HER 3 antibody or isotype control antibody for 1.5 hours at 4 ℃. The anti-HER 3 antibody clone LJM716 (e.g.described in Garner et al, cancer research (2013) 73: 6024-.

Cells were washed three times with FACS buffer (PBS containing 5mM EDTA and 0.5% BSA) at 2-8 ℃ and resuspended in FITC-conjugated anti-FC antibody (invitrogen, usa) for 40 minutes. The cells were washed again and resuspended in 200. mu. LFACS flow buffer (PBS with 5mM EDTA) for flow cytometry analysis using MACSQurant 10 (Meitian whirlpool Biotech, Germany). After collection, all raw data were analyzed using Flowlogic software. Cells were gated using forward and side scatter curves and the percentage of positive cells in native and over-expressed cell populations was determined.

The results are shown in fig. 1 to 4 and fig. 30 to 32. The anti-HER 3 antibody was shown to have very high specific binding to human HER 3. 10D1 and LJM716 have been shown to bind to a similar extent to cells expressing human HER 3.

3.2 ELISA assays for determining antibody specificity and Cross-reactivity

ELISA was used to determine the binding specificity of the antibodies. Binding of the antibody to the human HER3 polypeptide and to the respective mouse, rat and monkey homologues of HER3 was analysed (Sino bio ltd, china). Antibodies were also analyzed for their ability to bind to human EGFR and human HER2 (Sino bio-ltd, china).

ELISA was performed according to standard protocols. Briefly, prepared with Phosphate Buffered Saline (PBS)A96-well plate (Nonck, Denmark) was coated with 0.1. mu.g/ml of the polypeptide of interest and incubated at 4 ℃ for 16 hours. After blocking with 1% BSA in Tris Buffered Saline (TBS) for 1 hour at room temperature, the anti-HER 3 antibody was serially diluted to a maximum concentration of 10. mu.g/ml and added to the plates. After 1 hour incubation at room temperature, the plates were washed 3 times with TBS (TBS-T) containing 0.05% Tween 20, and then incubated with HRP-conjugated anti-His antibody (Life technologies, Inc., USA) for 1 hour at room temperature. After washing, the plates were developed with the

colorimetric detection substrate

3,3', 5,5' -tetramethylbenzidine (Turbo-TMB; pierce, USA) for 10 minutes. With 2M H ₂ SO ₄ The reaction was stopped and the OD was measured at 450 nM.

The results of ELISA are shown in fig. 5 to 7 and fig. 33.

The results show that anti-HER 3 antibody clone 10D1 did not bind to human HER2 or human EGFR even at high concentrations of antibody (fig. 5A). It was also found that anti-HER 3 antibody clone 10D1 showed significant cross-reactivity with mouse HER3, rat HER3 and cynomolgus monkey HER3 (fig. 5B).

The results indicate that anti-HER 3 antibody clone 4-35-B2 binds to human HER2 and human EGFR (fig. 6A). anti-HER 3 antibody clone 4-35-B2 also showed significant cross-reactivity with mouse HER3, rat HER3, and cynomolgus monkey HER3 (fig. 6B).

The results indicate that anti-HER 3 antibody clone 4-35-B4 binds to human HER2 and human EGFR (fig. 7A). anti-HER 3 antibody clone 4-35-B4 also showed significant cross-reactivity with mouse HER3, rat HER3, and cynomolgus monkey HER3 (fig. 7B).

All 10D1 variants were demonstrated to bind to human HER3 (fig. 33A and 33B).

3.3 Overall affinity Studies Using the Octet QK384 System

The binding affinity of the anti-HER 3 antibody clone in the form of IgG1 to human HER3 was analyzed.

Biolayer interferometry (BLI) experiments were performed using an Octet QK384 system (ford bio). Anti-human IgG capture (AHC) octagon sensor probe (pallford bio, usa) was used to capture anti-HER 3 antibody (25 nM). All measurements were made at 25 ℃ at a speed of 1000 rpm. By applying bands of different concentrations Hi s-tagged human HER3 antigen was loaded for 120s, and kinetic measurements of antigen binding were then performed by transferring the biosensor into assay buffer containing wells for dissociation for 120 s. Sensorgrams were referenced to buffer effects and then fitted using Octet QK384 user software (pallford bio, usa). Overall fitting of kinetic responses using a site binding model to obtain binding values (K) _on ) Dissociation rate (K) _off ) Rate constant and equilibrium dissociation constant (K) _D ). Only reliably by software (R) ² >0.90) fitted curve was included in the analysis.

FIG. 8 shows a representative sensorgram for analysis of clone 10D 1. Affinity K for clone 10D1 found to bind to human HER3 _D ＝9.58nM。

The humanized/optimized 10D1 variant binds human HER3 with very high affinity. Representative sensorgrams are shown in fig. 36A to 36M.

The affinity determined for the 10D1 clone variant is shown below:

antibody cloning	Affinity (K) _D )
		10D1_c89	72.6pM
10D1_c90	<1pM
		10D1_c91	176pM
10D1_11B	0.41nM
		10D1_c85o	17.3nM
10D1_c87	<1pM
		10D1_c93	<1pM
10D1_c76	<1pM
		10D1_c77	1.93nM
10D1_c78	<1pM
		10D1_c75	<1pM
10D1_c85	7.58nM
		10D1_c85o1	18.2nM

As a result, it was found that clone 4-35-B2 was represented by K _D Binding to human HER3 with an affinity of 80.9nM (fig. 9), clone 4-35-B4 with K _D Binding to human HER3 with an affinity of 50.3nM (fig. 10).

3.4 analysis of thermal stability by differential scanning fluorescence

Briefly, three 0.2mg/mL reaction mixtures of antibody and ruby orange dye (sequoyield) were prepared in 25 μ LPBS, transferred to reaction wells of a microampere optical 96-well plate (sequoyield), and sealed with microampere optical film (sequoyield). The melting curve was run in 7500 Rapid real-time PCR System (applied biosystems), TAMRA was chosen as reporter and ROX as reference fluorescence. The thermal profile comprises an initial step at 25 ℃ for 2 minutes and a final step at 99 ℃ for 2 minutes, with a ramp rate of 1.2%. The first derivative of the raw data is plotted as a function of temperature to obtain a derivative melting curve. The melting temperature (Tm) of the antibody was extracted from the peak of the derivative curve.

The first derivative of the raw data obtained by differential scanning fluorescence analysis of the thermostability of antibody clone 10D1 is shown in figure 11. Three different samples of antibody were analyzed. The Tm was determined to be 70.3 ℃.

The 10D1 variant clone and LJM716 were also analyzed. The first derivative of the raw data and the determined Tm are shown in fig. 35A to 35C.

3.5 anti-HER 3 antibody 10D1 epitope analysis

anti-HER 3 antibody 10D1 was analyzed to determine whether it competed with anti-HER 3 antibody MM-121 and/or LJM-716 for binding to HER 3. The epitope for MM-121 has been mapped to domain I of HER 3; it blocks the NRG ligand binding site. The epitope of LJM-716 has been mapped to conformational epitopes distributed in domains II and IV and locks HER3 in the inactive conformation.

Biolayer interferometry (BLI) experiments were performed using the Octet QK384 system (ford bio). A His-tagged human HER3(75 nM; 300s) was captured using an anti-five HIS (HIS1K) coated biosensor probe (Flter Bio Inc., USA). Binding to saturating antibodies was detected (400 nM; 600s) followed by a dissociation step (120s), followed by detection of binding to competing antibodies (300 nM; 300s) followed by a dissociation step (120 s). The variable region of the MM-121 antibody was cloned into a PDZ vector with a human IgG2 and Ig κ Fc backbone. The variable region of the LJM-716 antibody was cloned into a PDZ vector with human IgG1 and Ig κ Fc backbone.

The analysis results are shown in fig. 14A and 14B. The anti-HER 3 antibody was found not to compete with MM-121 and/or LJM-716 for binding to HER 3.

10D1 was found to bind to a unique and topologically remote epitope of HER3 compared to MM-121 and/or LJM-716.

The epitope of 10D1 was located using 15 amino acids that overlap to cover the entire HER3 extracellular domain. Each unique 15 residues were extended at the C and N ends by GS linkers and coupled to a unique well in a 384 well plate, followed by incubation in 0.1, 1, 10 and 100ug/ml 10D1 antibody for 16 hours at 4 ℃. The plates were washed and then incubated with POD conjugated goat anti-human IgG for 1 hour at 20 ℃. Finally, the POD substrate solution was added to the wells for 20 minutes. Prior to conjugation, evaluation was performed by measuring chemiluminescence at 425nm using a LI-COR Odyssey imaging system, followed by quantification and analysis using the PepSlide analysis software package. The experiment was performed twice.

The 10D1 epitope was found not directly on the β -hairpin structure of the HER3 dimerization arm located at domain II, but at the N-terminal dimerization interface of the β -hairpin.

Determining that the HER3 site bound to 10D1 and 10D1 derived clones corresponds to binding to positions 218 to 235 of the amino acid sequence of human HER3 (e.g., as shown in SEQ ID NO: 1); the amino acid sequence of this region of HER3 is shown in SEQ ID NO: 229. within this region, two consensus binding site motifs were identified and are shown in SEQ ID NO: 230 and 231.

Binding to this position of HER3 functions to block HER family heterodimerization and subsequent downstream signaling pathways (see example 4). Binding was ligand (NRG) independent. The 10D1 binding site is solvent accessible in both the open and closed conformation of HER3, is not conserved between HER3 and other HER family members, and is 100% conserved in the human, mouse, rat, and monkey HER3 orthologs.

Example 4: functional characterization

4.1 inhibition of dimerization of HER2 and HER3

The ability of anti-HER 3 antibodies to inhibit HER3 and HER2 heterodimerization was analyzed.

Briefly, 96-well plates (Nonck, Denmark) were coated with 0.1. mu.g/ml His-tagged HER2 protein in PBS for 16h at 4 ℃. 1% BSA at room temperatureAfter 1 hour blocking with PBS, recombinant biotinylated human HER3 protein was added in the presence of different concentrations of anti-HER 3 antibody clone 10D1 and the plates were incubated at room temperature for 1 hour. The plates were then washed 3 times and then incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. After washing, the plates were developed with the

colorimetric detection substrate

3,3',5,5' -tetramethylbenzidine (Turbo-TMB; pierce, USA) for 10 minutes. With 2M H ₂ SO ₄ The reaction was stopped and the OD was measured at 450 nM.

The results are shown in FIG. 15. The anti-HER 3 antibody clone 10D1 was found to inhibit the interaction between HER2 and HER3 in a dose-dependent manner.

In further experiments, inhibition of HER2: HER3 dimerization was analyzed.

In further experiments, HER2 was evaluated using the PathHunter pertuzumab bioassay kit (discover x) according to the manufacturer's instructions: inhibition of HER3 dimerization.

Briefly, HER2 and HER3 overexpressing U2OS cells were thawed using 1ml of pre-warmed CP5 medium, seeded with 5,000 cells per well, and cultured at 5% CO ₂ The culture was carried out at 37 ℃ for 4 hours under an atmosphere. Cells were then treated with 8-point serial dilutions of 10d1f. fca or pertuzumab starting at 25 μ g/ml.

After 4 hours of incubation, 30ng/ml of regulatory protein- β 2 was added to each well and the cells were incubated for a further 16 hours. 10 μ L PathHunter bioassay detection reagent 1 was added to the wells and incubated for 15 minutes at room temperature in the dark. Subsequently 40 μ L PathHunter bioassay detection reagent 2 was added and incubated in the dark at room temperature for 60 minutes. The plate was then read by Synergy4 Biotek after 1 second.

The results are shown in FIG. 65. Fca was found to have higher inhibition of HER2 than pertuzumab: HER3 ability to dimerize as reflected by its lower IC 50.

4.2 identification of cancer cell lines for analysis

The inventors characterized the expression of EGFR protein family members in cancer cell lines to identify suitable cells to study HER3 inhibition.

FIG. 16A shows mRNA expression data for N87, SNU16, HT29, FaDu, A549, HCC95, OvCAR8 and AHCN cells for EGFR family members and ligands, as described in cancer cell line encyclopedia (CCLE; Barretina et al, Nature (2012) 483: 603-607. and cancer cell line encyclopedia alliance, and genomics of drug sensitivity in cancer alliance, Nature (2015) 528: 84-87). Figure 16A also shows protein expression data for EGFR, HER2, and HER3 as determined by FlowLogic.

The cell lines used in the experiments were purchased from ATCC and cultured as recommended. Briefly, cell lines were maintained in the indicated cell culture media and supplemented with 10% FBS and 1% Pen/Strep. Cells were cultured at 37 ℃ in a 5% CO2 incubator. Cultured cells were seeded in 96-well plates at the appropriate seeding density: HT29, HCC95, FADU and OvCar8 cells were seeded at 2000 cells/well, NCl-N87 cells at 5000 cells/well, SNU-16, ACHN and cells at 1500 cells/well, A549 cells at 1200 cells/well.

Figure 16B shows surface expression of EGFR, HER2, and HER3 as determined by flow cytometry. Briefly, 500,000 cells were stained with primary antibody (20. mu.g/ml) in staining buffer containing 0.5% BSA and 2mM EDTA for 1.5h at 4 ℃. Anti-human Alexafluor488 was used as a secondary antibody and treated at 10. mu.g/ml for 20 minutes at 4 ℃.

4.3 inhibition of HER 3-mediated Signal transduction

The ability of anti-HER 3 antibody 10D1 to inhibit HER-3 mediated signaling in vitro was analyzed.

Briefly, 5% CO at 37 deg.C ₂ N87 and FaDu cells were seeded into wells of a 6-well plate containing 10% serum. After 16 hours, cells were starved in cell culture medium containing 1% FBS overnight (to reduce growth factor-induced signaling in serum). On the following day, cells were treated with 50 μ g/ml anti-HER 3 antibody 10D1 for 4 hours, followed by 15 minutes of stimulation with NRG (100 ng/ml). The proteins were then extracted, quantified using a standard Bradford protein assay, separated by SDS-PAGE, and transferred to nitrocellulose membranes. The membrane was then blocked and washed with anti-pHER 3, anti-pAK at 4 ℃T, pan-anti-HER 3, pan-anti-AKT, and anti- β -actin antibodies were immunoblotted overnight. Blots were visualized by Bio-Rad Clarity Western ECL substrate and bands were quantified using densitometry; data were normalized to the beta actin control.

The results are shown in FIG. 17. The anti-HER 3 antibody 10D1 was found to inhibit HER3 phosphorylation and downstream signaling.

In further experiments, the inventors investigated the intracellular signaling pathway affected by HER3 inhibition mediated by an anti-HER 3 antibody.

FaDu cells were incubated at 37 ℃ with 5% CO ₂ The wells of a 6-well plate were inoculated with a serum concentration of 10%. After 16 hours, cells were cultured by starvation overnight in 1% FBS cell culture medium. On the following day, cells were treated with 50 μ g/ml anti-HER 3 antibody 10D1 for 4 hours, followed by 15 minutes of stimulation with NRG (100 ng/ml). The proteins were then extracted, quantified using a standard Bradford protein assay, and incubated with pre-blocked phosphoprotein antibody array membranes (leisochnology) overnight at 4 ℃. The membrane was then washed with wash buffer and incubated with the detection antibody mixture for 2 hours at room temperature, then washed and incubated with HRP-conjugated anti-IgG. After 2 hours, the membrane was washed and probed with the kit detection buffer. Images were captured with Syngene Gbox imaging system, the intensity of each spot/phosphoprotein was measured, and percent inhibition was calculated by comparison with the intensity of cells treated in the same manner without antibody.

The results are shown in FIG. 18. The anti-HER 3 antibody 10D1 was found to inhibit PI3K/AKT/mTOR and MAPK signaling.

In further experiments, the inventors investigated the effect of anti-HER 3 antibody 10D1 treatment on proliferation of cells expressing HER 3.

Briefly, N87 and FaDu cells were treated with a 9-point semilog dilution starting at 100 μ g/ml with serial dilutions of anti-HER 3 antibody 10D 1. After 5 days, cell proliferation was measured using the CCK-8 proliferation assay (Homon chemical, Japan) according to the manufacturer's instructions. Briefly, 1x CCK-8 solution was added to each well, followed by incubation at 37 ℃ for 2 hours. The OD was then measured at 450 nm.

Figures 19A and 19B show the percent cell fusion relative to untreated control cells (data points are the average of three replicates).

The anti-HER 3 antibody 10D1 showed dose-dependent inhibition of cell proliferation on N87 cells and FaDu cells.

Example 5: in vivo analysis

5.1 pharmacokinetic analysis

Female NCr nude mice aged about 6-8 weeks were housed under specific pathogen-free conditions and treated according to Institutional Animal Care and Use Committee (IACUC) guidelines.

500 μ g of anti-HER 3 antibody was administered and blood was obtained from 3 mice by cardiac puncture at baseline (-2 hours), 0.5 hours, 6 hours, 24 hours, 96 hours, 168 hours and 336 hours after administration. Antibodies in serum were quantified by ELISA.

The results are shown in FIG. 50. The half-life of anti-HER 3 antibody clone 10D1 was found to be 16.3 days in NCr nude mice.

5.2 safe immunotoxicity

In silico analysis of HER3 antibody clone 10D1 was performed using IMGT DomainGapAlign (Ehrenmann et al, nucleic acid research, 38, D301-307(2010)) and IEDB deimmunization tool (Dhanda et al, immunology (2018)153 (1): 118-.

The number of potentially immunogenic peptides of anti-HER 3 antibody clone 10D1 was small enough to be considered safe and did not have any other properties that could lead to potential exploitability problems.

Figure 37 is a table summarizing the characteristics of 10D1 variant clones with respect to safety and developability.

Mice treated with anti-HER 3 antibody in the experiment described in example 5.3 were monitored for changes in body weight and gross anatomy. No differences were detected in these mice compared to mice treated with vehicle alone.

Hematological toxicity was studied in an experiment in which the disease was 6-8 weeks oldFemale BALB/c mice (20-25g) were injected intraperitoneally with a single dose of 1000. mu.g of anti-HER 310D 1 antibody or an equal volume of PBS. Blood samples were obtained 96 hours after injection and were obtained by flow cytometry and NA ⁺ 、K ⁺ And Cl ^- The number of different types of leukocytes is analyzed.

Fig. 51A and 51B show that the number of different cell types and electrolyte indices were found to be within the charles river reference range (3 mice), with no significant difference from the PBS treated group (3 mice). The left bar represents the vector and the right bar represents the 10D1 treatment, with dotted lines indicating the end of the Charles River (Charles River) reference range. No clinical signs, gross anatomy or body weight differences were found between the different groups.

The mice were also analyzed for correlation of hepatotoxicity, nephrotoxicity and pancreatic toxicity 96 hours after injection. After administration of a single dose of 1000 μ g of anti-her 3 antibody, the levels of alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), Blood Urea Nitrogen (BUN), Creatinine (CREA), alkaline phosphatase (ALP), Glucose (GLU), Calcium (CAL), total Bilirubin (BIL), Total Protein (TPR), and Albumin (ALB) detected were found to be not significantly different in the charles river reference range compared to the PBS treated group. These are shown in fig. 51C to 51F. The left bar represents the vector and the right bar represents the 10D1 treatment, with dotted lines indicating the end of the Charles River (Charles River) reference range. 10D1 treatment had no effect on renal, hepatic or pancreatic index and therefore did not affect normal renal, hepatic or pancreatic function.

5.3 in vivo therapeutic efficacy analysis of cancer

Female NCr nude mice, about 6-8 weeks old, were purchased from InVivos (singapore). Animals were housed in specific pathogen-free conditions and cultured according to Institutional Animal Care and Use Committee (IACUC) guidelines.

Cell lines used included N87 cells (gastric cancer), FaDu cells (head and neck cancer), OvCAR8 cells (ovarian cancer), SNU16 cells (gastric cancer), HT29 cells (colorectal cancer), a549 cells (lung cancer), HCC95 cells (lung cancer) and AHCN cells (kidney).

Tumor volumes were measured 3 times per week using a digital caliper and calculated using the formula [ L × W2/2 ]. Once the tumor length of the control arm >1.5cm, the study endpoint was considered to have been reached.

5.3.1N87 model

FIG. 20 shows the study of anti-HER 3 antibody 10D1 in the N87 cell line derived from a mouse gastric cancer model (example 2.2 [ 1)]) Experimental results of anticancer effect of (1). By mixing 1X 10 ⁶ N87 cells were injected subcutaneously into the right flank (N ═ 6 mice per treatment group) to establish a model.

Administered intraperitoneally at a dose of 500 μ g (10 doses) once every two weeks for 10D 1; control treatment groups received an equal volume of PBS.

The anti-HER 3 antibody clone 10D1 was found to be very effective in this model and was able to inhibit tumor growth by about 76%.

FIG. 21 shows the results obtained in a similar experiment, in which anti-HER 3 antibody clone 4-35-B4 was administered intraperitoneally at a dose of 11mg/kg weekly (4 doses total). The anti-HER 3 antibody clone 4-35-B4 was found to be very effective in this model and was able to inhibit tumor growth by approximately 60%.

5.3.2SNU16 model

FIG. 22 shows the study of anti-HER 3 antibody 10D1 (example 2.2 [1 ] of SNU16 cell line-derived mouse gastric cancer model]) Experimental results on anticancer effects of (1). By subcutaneous injection of 1X 10 to the right flank ⁶ SNU16 cells were modeled (n-6 mice per treatment group).

Every two weeks, 500 μ g (9 doses) of each dose, 10D1 was administered intraperitoneally; control treatment groups received an equal volume of PBS.

anti-HER 3 antibody clone 10D1 was found to be very effective in this model and was able to inhibit tumor growth by approximately 68%.

5.3.3FaDu model

FIG. 23 shows the study of anti-HER 3 antibody 10D1 in a mouse model of head and neck squamous cell carcinoma derived from the FaDu cell line (example 2.2 [1 of]) Experimental results of anticancer effect of (1). By mixing 1X 10 ⁶ FaDu cells were injected subcutaneously into the right flank of female NPG mice to establish a model(NOD scid γ phenotype; n ═ 6 mice per treatment group).

10D1 was administered intraperitoneally at 500 μ g/week (4 doses total). Control treatment groups received an equal volume of PBS or an equal dose of isotype control antibody.

The anti-HER 3 antibody clone 10D1 was found to be very effective in this model and was able to inhibit tumor growth by approximately 85%.

FIG. 24 shows the study of anti-HER 3 antibody 10D1 in a mouse model of FaDu cell line derived squamous cell carcinoma of head and neck (example 2.2 [ 1)]) Experimental results on anticancer effects of (1). By mixing 1 × 10 ⁶ FaDu cells were injected subcutaneously into the right flank of female NCr nude mice (n ═ 6 mice per treatment group) to model.

Administered intraperitoneally at a dose of 500 μ g (8 doses) once every two weeks at 10D 1; control treatment groups received an equal volume of PBS.

The anti-HER 3 antibody clone 10D1 was found to be very effective in this model and was able to inhibit tumor growth by about 86%.

5.3.4OvCAR8 model

FIG. 25 shows the study of anti-HER 3 antibody 10D1 in a mouse model of ovarian cancer derived from the OvCAR8 cell line (example 2.2 [ 1)]) Experimental results of anticancer effect of (1). By mixing 1 × 10 ⁶ OvCAR8 cells were injected subcutaneously into the right flank of female NCr nude mice to establish a model (6 mice per treatment group).

anti-HER 3 antibody clone 10D1 was found to be very effective in this model and was able to inhibit tumor growth by only 74%.

5.3.5HCC-95 model

FIG. 26 shows that anti-HER 3 antibody 10D1 (example 2.2 [1 ] of HCC-95 cell line-derived squamous cell lung carcinoma mouse model]) Experimental results on anticancer effects of (1). By mixing 1 × 10 ⁶ HCC-95 cells were subcutaneously injected into the right flank of female NCr nude mice to establish a model (n ═ 6 mice per treatment group).

Administered intraperitoneally at a dose of 500 μ g (4 doses) once every two weeks at 10D 1; control treatment groups received an equal volume of PBS.

The anti-HER 3 antibody clone 10D1 was found to be very effective in this model and was able to inhibit tumor growth by approximately 90%.

5.3.6A549 model

FIG. 27 shows that the anti-HER 3 antibody 10D1 (example 2.2 [1 ] of]) Experimental results on anticancer effects of (1). By mixing 1 × 10 ⁶ A549 cells were subcutaneously injected into the right flank of female NCr nude mice to establish a model (n ═ 6 mice per treatment group).

The anti-HER 3 antibody clone 10D1 was found to be very effective in this model and was able to inhibit tumor growth by only 91%.

FIG. 28 shows the results obtained in a similar experiment, in which 1X 10 was injected ventrally, on the right, by injecting NPG female mice (NOD scid gamma phenotype) ⁶ In an A549 cell line derivative model established by the A549 cells, the anti-cancer effect of the anti-HER 3 antibody clone 4-35-B2 is researched. anti-HER 3 antibody clone 4-35-B2 was administered intraperitoneally weekly at 500 μ g per dose (total of 4 doses). The control treated group received an equal volume of PBS (6 mice per treated group).

Also, the anti-HER 3 antibody clone 4-35-B2 was found to have high potency in this model and was able to inhibit tumor growth by about 63%.

5.3.6ACHN model

FIG. 29 shows the study of anti-HER 3 antibody 10D1 (example 2.2 [1 ] of [1 ] in a mouse model of ACHN cell line-derived renal cell carcinoma]) Experimental results on anticancer effects of (1). By mixing 1X 10 ⁶ ACHN cells were subcutaneously injected into the right flank of female NCr nude mice to establish a model (n ═ 6 mice per treatment group).

Administered intraperitoneally at a dose of 500 μ g (7 doses) once every two weeks at 10D 1; control treatment groups received an equal volume of PBS.

The anti-HER 3 antibody clone 10D1 was found to be very effective in this model and was able to inhibit tumor growth by about 61%.

5.4 treatment of gastric cancer

First in humans

HER2+ advanced gastric cancer patients who failed or failed trastuzumab therapy can be treated by intravenous injection of an anti-HER 3 antibody selected from the group consisting of: 10D1, 10D1_ c75, 10D1_ c76, 10D1_ c77, 10D1_ c78v1, 10D1_ c78v2, 10D1_11B, 10D1_ c85v1, 10D1_ c85v2, 10D1_ c85o1, 10D1_ c85o2, 10D1_ c87, 10D1_ c89, 10D1_ c90, 10D1_ c91, 10D1_ c92 and 10D1_ c93, the dose calculated according to the safety adjusted "lowest expected bioeffect level" (MABEL) method. Patients were monitored 28 days after dosing.

Patients were then evaluated according to the general terminology criteria for adverse events (CTCAE) to determine the safety and tolerability of the treatment, as well as to determine the pharmacokinetics of the molecule.

Treatment with the anti-HER 3 antibody was found to be safe and tolerable.

Dose escalation-monotherapy

Treating 12-48 HER2+ advanced gastric cancer patients who failed or failed to receive trastuzumab by intravenous injection of an anti-HER 3 antibody selected from the group consisting of: 10D1, 10D1_ c75, 10D1_ c76, 10D1_ c77, 10D1_ c78v1, 10D1_ c78v2, 10D1_11B, 10D1_ c85v1, 10D1_ c85v2, 10D1_ c85o1, 10D1_ c85o2, 10D1_ c87, 10D1_ c89, 10D1_ c90, 10D1_ c91, 10D1_ c92, and 10D1_ c93 (e.g., 10D1_ c89, 10D1_ c90, or 10D1_ c 91; e.g., 10D1_ c89), dose escalation design incremental dose (EWOC) based on the 3+3 model.

Patients were then evaluated according to the general terminology criteria for adverse events (CTCAE) to determine the safety and tolerability of the treatment, and to assess the pharmacokinetics of the molecule and the efficacy of the treatment. The Maximum Tolerated Dose (MTD) and the Maximum Administered Dose (MAD) were also determined.

Dose escalation-combination therapy

Treating 9-18 failed or trastuzumab HER2+ advanced gastric cancer patients by intravenous injection of an anti-HER 3 antibody selected from: 10D1, 10D1_ c75, 10D1_ c76, 10D1_ c77, 10D1_ c78v1, 10D1_ c78v2, 10D1_11B, 10D1_ c85v1, 10D1_ c85v2, 10D1_ c85o1, 10D1_ c 1 (e.g. 10D1_ c 1, 10D1_ c 1 or 10D1_ c 1; e.g. 10D1_ c 1) in combination with trastuzumab, increasing amounts of anti-PD antibodies (3mg/kg) based on 3+3 model.

Patients were then evaluated according to the general terminology criteria for adverse events (CTCAE) to determine the safety and tolerability of the treatment, and to assess the pharmacokinetics and therapeutic efficacy of the molecule.

Dose escalation

HER2+ advanced gastric cancer patients who have failed recent trastuzumab therapy, whose tumors are well characterized genetically and histologically, can be treated with an anti-HER 3 antibody selected from: 10D1, 10D1_ c75, 10D1_ c76, 10D1_ c77, 10D1_ c78v1, 10D1_ c78v2, 10D1_11B, 10D1_ c85v1, 10D1_ c85v2, 10D1_ c85o1, 10D1_ c85o2, 10D 2 _ c 2 (e.g. 10D 2 _

c

2, 10D 2 _

c

2 or 10D 2 _ c 2) in combination with trastuzumab, cisplatin, and 5-FU or capecitabine (capecitabine).

The anti-HER 3 antibody was found to be safe and tolerable, and was able to reduce the number/proportion of cancer cells, reduce expression of tumor cell markers, increase progression-free survival and increase overall survival.

Example 6: affinity matured and humanized clones

Humanization of the parent mouse antibody 10D1P variable regions was accomplished by CDR grafting. The human framework sequences used for grafting were identified by aligning the parent amino acid sequences with the human V domain database and selecting the genes with the highest identity to the parent sequences. After grafting of the mouse CDRs into selected human frameworks, residues at canonical positions of the framework are re-mutated to the parental mouse sequence to retain antigen binding. 9 humanized variants of 10D1P were designed.

Using yeast display, affinity to human HER3 was increased by two rounds of affinity maturation. In the first round, a mixed library of 9 designed variants was constructed by random mutagenesis and screened by flow cytometry using biotinylated antigen. In the second round, one heavy and one light chain clone isolated in the first round was used as template to generate and screen another library. In total 10 humanized and affinity matured clones were isolated.

Potential features (immunogenicity, glycosylation sites, exposed reactive residues, aggregation potential) in the variable region of the designed and isolated 10D1P humanized variants were evaluated using in silico prediction tools. The sequences were deimmunized using the IEDB deimmunization tool. The final sequence of 10D1F was selected from the optimized variants based on its developability characteristics and in vitro physicochemical and functional properties.

Clone 10D1F contained SEQ ID NO: 36 and the VH of SEQ ID NO: 83 VL. 10D1F showed 89.9% homology to the human heavy chain and 85.3% homology to the human light chain.

An antigen binding molecule comprising the 10D1F variable region and the human IgG1 constant region, consisting of the polypeptides shown in SEQ ID NOS: 206 and 207, labeled 10D1F. FcA (sometimes also referred to herein as "10 D1F. A" or "anti-HER 3 clone 10D1_ c89 IgG 1" -see, e.g., [16] of example 2.2).

Example 7: fc engineering

The 10D1 and 10D1 variants were engineered to include mutations in the CH2 and/or CH3 regions to increase antibody potency, e.g., optimize Fc effector function, enhance antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP) and improve half-life.

Fca was modified to include "GASDALIE" (G236A, S239D, a330L, I332E) and "LCKC" (L242C, K334C) in the CH2 region by modifying the Fc regions of clones 10D1 and 10D1f. It was found experimentally that the substitution by GASDALIE increased the affinity for Fc γ riia (ga) and Fc γ riiia (sdaleie) receptors and enhanced ADCP and NK-mediated ADCC (see example 8.8), while decreasing the affinity for C1q (AL) and decreasing CDC. It was experimentally found that the substitution of LCKC increased the thermostability of the Fc region by creating new intramolecular disulfide bonds.

A modified version of the 10d1f. fca heavy chain polypeptide comprising GASDALIE and LCKC mutations is set forth in SEQ ID NO:225, respectively. The antigen binding molecule consisting of the polypeptides shown in SEQ ID NOs: 225 and 207 is labeled as 10D1F. FcB (sometimes also referred to herein as "10D 1F. B").

Modified versions of 10D1, containing GASDALIE and LCKC substitutions in the CH2 region, were prepared and analyzed for their ability to bind to the Fc receptor fcyriiia by biolayer interferometry. The 10D1VH-CH1-CH2-CH3 has the sequence shown in SEQ ID NO: 227, which contains GASDALIE and LCKC substitutions corresponding to G236A, S239D, a330L, I332E and L242C, K334C.

Briefly, a anti-five-HIS (HIS1K) coated biosensor probe (pallford bio, usa) was used to capture HIS-tagged Fc γ RIIIa (V158) (270nM) for 120 s. All measurements were made at 25 ℃ at a speed of 1000 rpm. The binding kinetics of antigen binding was measured by incubating the anti-her 3 antibody at various concentrations (500nM to 15.6nM) for 60s, and then transferring the biosensor to assay buffer containing pH 7.2 with a dissociation time of 120 s. Sensorgrams were referenced to buffer effects and then fitted using Octet QK384 user software (pallford bio, usa). Overall fitting of kinetic responses using a site binding model to obtain binding values (K) _on ) Dissociation rate (K) _off ) Rate constant and equilibrium dissociation constant (K) _D ). Only reliably by software (R) ² >0.90) fitted curve was included in the analysis.

The thermostability of the variants was also analyzed by differential scanning fluorescence analysis as described in example 3.4.

Fig. 38A and 38B show BLI analysis and thermal stability analysis, respectively, of 10D1 containing GASDALIE and LCKC Fc substitutions. The Fc engineered 10D1 variant showed significantly improved binding to Fc γ RIIIa (9-fold increase in affinity) compared to non-Fc engineered 10D1 (see figure 39A) and maintained thermostability above 60 ℃.

A 10D1 construct comprising a GASD substitution in the CH2 region was also prepared. The sequence of 10D1 VH-CH1-CH2-CH3 comprising the substitutions corresponding to G236A and S239D is shown in SEQ ID NO: 228.

the binding affinity of anti-HER 3 antibody clone 10D1 (example 2.2 [1]) and its GASD variant to Fc γ RIIIa was analyzed by biolayer interferometry. BLI proceeds as described above.

FIGS. 39A and 39B show representative sensorgrams, K _on 、K _off And K _D The value is obtained. As expected, the affinity of the 10D1 GASD variant (39B) for Fc γ RIIIa was greatly improved compared to 10D1 (39A).

The thermostability of the 10D1 GASD variant was also analyzed by differential scanning fluorescence analysis as described in example 3.4. The results are shown in FIG. 40.

Other 10D1F Fc variants

Another antibody variant was created which contained a N297Q substitution in the CH2 region. A representative sequence of 10D1F VH-CH1-CH2-CH3 comprising a N297Q substitution is shown in SEQ ID NO: 226. this silent form prevents both N-linked glycosylation of the Fc region and prevents Fc binding to Fc γ receptors and is used as a negative control.

Figures 41A and 41B show the binding affinity of 10D1F hIgG1 Fc variants 10D1f. fca and 10D1f. fcb to human and mouse Fc receptors, determined as described above. Fca was found to have significantly improved binding of 10d1f. fcb to human and mouse Fc γ and FcRn receptors compared to unmodified 10d1f. fca or commercial antibodies. ND-K due to undetermined Low binding affinity _D 。

Example 8: humanization and improved cloning Properties

8.1 analysis of cell surface antigen binding by flow cytometry

Wild Type (WT) HEK293 cells (which do not express high levels of HER3) and HEK293 cells transfected with vectors encoding human HER3 (i.e. HEK293 HER O/E cells) were incubated with 10 μ g/ml of the humanized anti-HER 3 antibody 10D1f. fca (10D1F), anti-HER 3 antibody 10D1(10D1P), or isotype control antibody for 1.5 hours at 4 ℃. The anti-HER 3 antibody clone LJM716 (e.g.Garner et al, cancer research (2013) 73: 6024-.

The cells were washed with buffer (PBS containing 2mM EDTA and 0.5% BSA) at 4 ℃ and resuspended in FITC-conjugated anti-FC antibody (Invitrogen, USA) at a concentration of 10. mu.g/ml for 20 minutes. The cells were washed again and resuspended in 200. mu.L of FACS flow buffer (PBS with 5mM EDTA) and analyzed by flow cytometry using MACSQurant 10 (Gentiando whirlpool Biotech, Germany). Unstained WT and transfected HEK293 cells were included in the assay as negative controls. After collection, all raw data were analyzed using Flowlogic software. Cells were gated using forward and side scatter curves and the percentage of positive cells in both native and over-expressed cell populations was determined.

The results are shown in FIGS. 42A and 42B. Fca binds to human HER3 with high specificity (42A) as anti-HER 3 antibody 10d1f. Fca, 10D1P and LJM716 bound to human HER3 expressing cells to a similar extent (42B).

8.2 ELISA assays for determination of antibody specificity and Cross-reactivity

Binding specificity of the 10d1f. fca antibody was confirmed by ELISA. The ability of the antibodies to bind to human HER3 polypeptide as well as human HER1(EGFR) and human HER2(Sino bio-ltd, china) was analyzed. Human IgG isotype and irrelevant antigen were included as negative controls.

ELISA was performed according to standard protocols. Plates were coated with 0.1. mu.g/ml of the polypeptide of interest in Phosphate Buffered Saline (PBS) for 16h at 4 ℃. After blocking with 1% BSA in Tris Buffered Saline (TBS) for 1 hour at room temperature, anti-HER 3 antibody was serially diluted to a maximum concentration of 10. mu.g/ml and added to the plate. After 1 hour incubation at room temperature, the plates were washed 3 times with TBS (TBS-T) containing 0.05% Tween 20, and then incubated with HRP-conjugated anti-His antibody (Life technologies, Inc., USA) for 1h at room temperature. After washing, the plate was developed with a

colorimetric detection substrate

3,3', 5,5' -tetramethylbenzidine (Turbo-TMB; pierce, USA) for 10 minutes. With 2M H ₂ SO ₄ The reaction was stopped and OD was measured at 450 nM.

The results are shown in FIG. 43. Fca was found not to bind human HER2 or human HER1(EGFR) even at high concentrations of antibody, anti-HER 3 antibody 10d1f.

Fca ability to bind HER4 was analyzed by flow cytometry at 10d1f. Wild Type (WT) HEK293 cells (which do not express high levels of HER4) and HEK293 cells transfected with vectors encoding human HER4 (i.e. HEK293 HER O/E cells) were incubated with 10 μ g/ml of anti-HER 3 antibody 10D1f. fca (10D1F) or isotype control antibody (negative control) for 1.5 hours at 4 ℃. The anti-HER 3 antibody clone LJM716 (e.g.described in Garner et al, cancer research (2013) 73: 6024-6035) and MM-121 (Selizumab) as described in example 3.5 were used as positive controls in the assay. Commercial anti-HER 4 antibody (Rofos, cat # FAB11311P) was also included. Unstained HEK293 cells were included as negative controls in the assay.

HEK293 cells were incubated with 10. mu.g/ml of each antibody for 1 hour at 4 ℃. Flow cytometry was performed as described above. Cells were exposed to FITC-conjugated anti-FC antibody (invitrogen, usa) for 30 min at 4 ℃.

The results are shown in FIG. 44. Fca was found not to bind cell surface expressed HER4, anti-HER 3 antibody 10d1f.

In addition, the ability of antibody 10d1f. fca to bind to HER3 polypeptide homologs of mouse, rat and monkey (Sino bio-inc., china) was analyzed. HER3 homologues of mouse (m.musculus), rat (r.norvegicus) and cynomolgus (m.cynomolgus) share 91.1, 91.0 and 98.9% sequence identity with human HER3, respectively, while the HER3 signaling pathway is conserved among these four species.

The ELISA was performed as described above.

The results are shown in FIG. 45. Fca antibody was found to have high affinity binding to cynomolgus monkey, mouse, rat HER3 and human orthologs, thus showing significant cross-reactivity between species.

8.3 full affinity Studies by Octet QK384 System

anti-HER 3 antibody clones 10d1f. fca and 10d1f. fcb were analyzed for binding affinity to human HER 3.

Biolayer interferometry (BLI) experiments were performed using an Octet QK384 system (ForteBio). Antibody (25 n)M) was coated onto an anti-human IgG capture (AHC) octagon sensor probe (pallford bio, usa). Binding was measured by titration of HIS-tagged human HER3 at baseline (60s), load (120s), baseline 2(60s), binding (120s), dissociation (FcA 120s, FcB 600s) and regeneration (15 seconds). Antigen concentrations are shown in the tables of fig. 46A and 46B. Sensorgrams were analyzed as described in example 3.3. Obtain the combination (K) _on ) Dissociation (K) _off ) Rate constant and equilibrium dissociation constant (K) _D ) The value of (c).

Representative sensorgrams for analysis of clones 10d1f. fca and 10d1f. fcb are shown in fig. 46A and 46B. FcA with K10D 1F _D High affinity binding to human HER3 at 72.6pM (46A). FcB with K10D 1F _D High affinity binding to human HER3 for 22.2pM (46B).

8.4 analysis of thermal stability by differential scanning fluorescence

Antibodies 10d1f. fca and 10d1f. fcb were subjected to differential scanning fluorescence as described in example 3.4.

The first derivative of the raw data obtained by analyzing the thermal stability of antibody clone 10d1f. fca by differential scanning fluorescence is shown in fig. 47A. Three different samples of antibody were analysed and the Tm determined to be 70.0 ℃.

The first derivative of the raw data obtained by differential scanning fluorescence analysis of the thermostability of antibody clone 10d1f. fcb is shown in fig. 47B. Three different samples of antibody were analysed and the Tm determined to be 62.7 ℃.

8.5 antibody purity assay

Antibodies 10d1f. fca and 10d1f. fcb were analyzed for purity by Size Exclusion Chromatography (SEC). Mu.g of 10D1F. FcA dissolved in 500. mu.l of PBS pH 7.2 or 150. mu.g of 10D1F. FcB dissolved in 500. mu.l of PBS pH 7.45 were injected into a Superdex 20010/30 GL column with PBS flow buffer at a flow rate of 0.75min/ml or 0.5min/ml, respectively, and the flux under A280 was recorded at room temperature.

The results are shown in fig. 48A (10d1f. fca) and 48B (10d1f. fcb).

8.6 anti-HER 3 antibody 10D1F. FcA epitope analysis

Fca was analyzed against HER3 antibody 10d1f to determine whether it competed with anti-HER 3 antibody M-05-74 or M-08-11 (roche) for binding to HER 3. The epitopes of M-05-74 and M-08-11 are both located in the beta hairpin structure of the HER3 dimeric arm located in domain II. M-08-11 does not bind to HER4, whereas M-05-74 recognizes the HER4 dimeric arm. M-05-74 and M-08-11 bind to HER3 independently of the ligand (NRG).

BLI experiments were performed as described in example 3.5, but with a little difference: 400nM competitor antibody was used. The variable regions of the M-05-74 and M-08-11 antibodies were cloned into PDZ vectors with human IgG1 and Ig kappa Fc backbones.

The analysis results are shown in FIGS. 49A and 49B. FcA antibodies were found not to compete with M-05-74 or M-08-11 for binding to HER 3. FcA binds a unique and topologically remote epitope of HER3 compared to M-05-74 and M-08-11. Fca binding to HER3 independent of ligand (NRG) 10d1f.

And (4) conclusion:

fca binding to human HER3 can be achieved in a ligand-independent manner.

FcA binding epitopes are distinct from the binding epitopes of M-05-74 and M-08-11 and are topologically distant.

8.7 inhibition of dimerization of HER2-HER3 and EGFR-HER3

Fca was analyzed for the ability of anti-HER 3 antibody 10d1f to inhibit heterodimerization of HER3 and HER 2.

Plate-based ELISA dimerization assays were performed according to standard protocols. Plates were coated with 1. mu.g/ml HER2-Fc protein. After blocking and washing, plates were incubated with different concentrations of candidate antibodies 10d1f. fca, MM-121, LJM716, pertuzumab, roche M05, roche M08 or isotype control and constant HER3 His2 μ g/ml and NRG 0.1 μ g/ml for 1 hour. The plates were then washed and incubated with anti-HIS HRP secondary antibody for 1 hour. The plates were washed, treated with TMB for 10 minutes, and washed with 2M H ₂ SO ₄ The reaction was stopped by terminating the solution. The absorbance was read at 450 nm.

The results are shown in FIG. 52. Fca was found to directly inhibit the interaction between HER2 and HER3 in a dose-dependent manner, as was anti-HER 3 antibody clone 10d1f.

In another assay, inhibition of dimerization is used

Pertuzumab

Bioassay kit (discovery x, san francisco, usa). HER2 and HER3 overexpressing U20S cells were thawed using 1ml of pre-warmed CP5 medium and 5% CO at 37 ℃ ₂ 5K cells were seeded for 4 hours separately. Fca, selitumumab or pertuzumab concentrations at 25 μ g/ml were serially diluted 10d1f. fca, and cells were treated with 8-point serial dilutions. After 4 hours of incubation, 30ng/ml of regulatory protein- β 2 was added to each well and the plates were further incubated for 16 hours. After incubation, 10 μ L PathHunter bioassay detection reagent 1 was added and incubated for 15 minutes in the dark at room temperature, then 40 μ L PathHunter bioassay detection reagent 2 was added, which was then incubated for 60 minutes at room temperature in the dark. The plates were read by Synergy4 Biotek after 1 second.

FcA was found to inhibit EC of HER2-HER3 heterodimerization ₅₀ The value was 3.715 e-11. In the same experiment, the EC of Selizumab/MM-121 was found ₅₀ Relative values are 6.788e-10, and EC for pertuzumab ₅₀ The relative value is 2.481 e-10.

Fca was analyzed for its ability to inhibit heterodimerization of EGFR and HER3 by the anti-HER 3 antibody 10d1f.

Plate-based ELISA dimerization assays were performed according to standard protocols. Plates were coated with 1. mu.g/ml human EGFR-His. After blocking and washing, plates were incubated with different concentrations of candidate antibodies 10d1f. fca, MM-121, LJM716, pertuzumab or isotype control and constant HER 3-biotin 4 μ g/ml and NRG 0.1 μ g/ml for 1 hour. The plates were then washed and incubated with anti-avidin HRP secondary antibody for 1 hour. The plates were washed, treated with TMB for 10 minutes, and treated with 2M H ₂ SO ₄ The reaction was stopped by terminating the solution. The absorbance was read at 450 nm.

The results are shown in FIG. 53. Fca was found to directly inhibit the interaction between EGFR and HER3 in a dose dependent manner, by the anti-HER 3 antibody clone 10d1f.

8.8 ability to induce ADCCAnalysis of

anti-HER 3 antibody clones 10d1f. fca and 10d1f. fcb were analyzed for their ability to induce antibody-dependent cellular cytotoxicity (ADCC).

Target cells (HEK 293 overexpressing HER 3) were plated at a density of 20,000 cells/well in U-bottom 96-well plates. Cells were treated with or without treatment with one of the following serially diluted (50,000 ng/ml-0.18 ng/ml) 10D1F. FcA, 10D1F. FcB, 10D1F. FcA _ N297Q (silent form), LJM-716, Serratizumab (MM-121), then at 37 ℃ and 5% CO ₂ And incubated for 30 minutes. Effector cells (human natural killer cell line No-GFP-CD16. NK-92; 176V) were added to the plates containing the target cells at a density of 60,000 cells/well.

The following controls were included: maximum LDH release by target cells (target cells only), spontaneous release (target cells and effector cells without antibody), background (medium only). Centrifuging the plates and mixing at 37 ℃ and 5% CO ₂ The mixture was incubated for 21 hours.

LDH release assay (Pierce LDH cytotoxicity assay kit): prior to assay, 10. mu.L of lysis buffer (10X) was added to the target cell maximum LDH release control and incubated at 37 ℃ and 5% CO ₂ Incubate for 20 minutes. After incubation, the plates were briefly centrifuged and 50 μ Ι _ of supernatant was transferred to a clear flat-bottom 96-well plate. The reaction was started by adding 50 μ L of LDH assay mixture containing substrate to the supernatant and incubating for 30 min at 37 ℃. The reaction was stopped by adding 50. mu.L of stop solution and the absorbance was recorded at 490nm and 680nm with a BioTek Synergy HT plate reader.

For data analysis, the absorbance from the test sample was corrected for background and spontaneous release from target and effector cells. The percent cytotoxicity of the test samples was calculated relative to the target cell maximum LDH release control and plotted as a function of antibody concentration.

The results are shown in FIG. 54. Fcb was found to induce potent ADCC activity of anti-HER 3 overexpressing cells in a dose dependent manner, anti-HER 3 antibody 10d1f.

8.9 inhibition of HER3 mediated Signal transduction

Fca was analyzed for the ability of the anti-HER 3 antibody 10d1f to inhibit HER-3 mediated signaling in vitro cancer cell lines.

N87, FaDu or OvCAR8 cells were seeded in wells of 6% well plates in 10% serum at 37 ℃ with 5% CO ₂ Overnight. Cells were starved for 16 hours with 0.2% FBS medium and then treated with different antibodies corresponding to cell line IC50 for 0.5 hours. The antibodies tested were: fca (10D1), Selitumumab (SBT), eggeminmumab (LJM), Pertuzumab (PTM), Cetuximab (CTX) and Trastuzumab (TZ).

Prior to harvest, cells were stimulated with 100ng/ml NRG 1. Proteins extracted from the cell lines were quantified using a standard Bradford protein assay. Protein samples (50. mu.g) were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membrane was then blocked with the indicated antibodies and immunoblotted. Results were visualized by Bio-Rad Clarity Western ECL substrate. Blots were quantified by densitometry and data normalized to β actin.

The results are shown in FIGS. 55A to 55C. Fca was found to inhibit HER3 phosphorylation and downstream signaling in N87(55A), FaDu (55B), OvCar8(55C) and a549(55D) cell lines against HER3 antibody 10D1f.

For experiments using N87 cells, a549 cells, OvCar8 cells, and FaDu cells, total RNA 16 hours after antibody treatment was analyzed and pathway activation was determined based on expression levels of key signal transduction pathway proteins by gene set enrichment analysis.

The analysis results are shown in FIGS. 63A to 63D. Fca is the most potent inhibitor of downstream signaling.

In further experiments using a549 cells, in vitro phosphorylation assays were performed as described above, except that the cells were treated with different antibodies for 0.5 hours or 4 hours. The results are shown in FIG. 64.

8.10 analysis of binding of antibodies provided in "open" and "closed" conformations to HER3

In further experiments, anti-HER 3 antibodies were analyzed to determine the affinity of binding to human HER3 in the context of human HER3: human NRG1 complex (i.e., HER3 is provided in an "open" conformation to ligand binding) compared to the affinity of binding to human HER3 in the absence of NRG1 complex (i.e., HER3 is provided in an "closed" conformation to no binding).

The binding of anti-HER 3 antibody to human HER3 was evaluated by BLI method. Briefly, an anti-human IgG capture (AHC) sensor (ForteBio) was loaded with anti-HER 3 IgG antibody (25 nM). Kinetic measurements were performed in the absence or presence of NRG 1. NRG1 and HER3 react at a 1: 1, wherein the complex is allowed to form at room temperature for 2 hours. His-tagged human HER3 or HER3-NRG1 complex was loaded on antibody-coated AHC sensors at different concentrations for 120s followed by a dissociation time of 120 s. All measurements were carried out at 25 ℃ at a rotation speed of 1000 rpm. The buffering effect of the sensor map was referenced and then analyzed using Octet QK384 software (ForteBio). The kinetic responses were fit to the whole using a site binding model to obtain binding values (K) _on ) Dissociation rate (K) _off ) Rate constant and equilibrium dissociation constant (K) _D )。

The following anti-HER 3 antibodies were analyzed in the experiment: 10D1F. A (example 2.2[16]), MM-121 and LJM-716.

The results are shown in FIGS. 78A to 78F. 10d1f.a was found to bind human HER3 with sub-picomolar affinity in the presence or absence of NRG1 (fig. 78A and 78B). MM-121 binds nanomolar to human HER3 in the presence or absence of NRG1 (fig. 78C and 78D). LJM-716 bound to human HER3 with subpicomolar affinity in the absence of NRG1 (fig. 78E). However, binding to HER3 was significantly reduced in the presence of NRG1 (fig. 78F).

8.11 conclusion

In summary, the results identified 10D1F as an anti-HER 3 antibody that binds to an epitope of HER3, providing it with a unique combination of properties, namely (i) it competitively inhibits heterodimerization of HER3 with EGFR or HER2 (see example 8.7 and figures 52 and 53), and (ii) binds similarly well to HER3 in the presence or absence of NRG1 (figures 78A and 78B).

Example 9: in vitro and in vivo analysis of humanized and modified clones

9.1 pharmacokinetic analysis

Mouse

500 μ g of anti-HER 3 antibody 10d1f. fca or 10d1f. fcb was administered and blood was obtained from 4 mice by cardiac puncture at baseline (-2 hours), 6 hours, 24 hours, 96 hours, 168 hours and 336 hours after administration. Antibodies in serum were quantified by ELISA.

Parameters for pharmacokinetic analysis were derived from non-compartmental models: maximum concentration (C) _max ) AUC (0-336hr), AUC (0-infinity), half-life (t) _1/2 ) Clearance (CL), dispensing quantity at steady state (V) _ss )。

The results are shown in FIGS. 56A and 56B. The half-life of anti-HER 3 antibody clone 10d1f. fca was found to be 253 hours (56A) and the half-life of anti-HER 3 antibody clone 10d1f. fcb was found to be 273 hours (56B) in NCr nude mice.

Rat

The 10D1F variant was analyzed to determine the single dose pharmacokinetic profile of female Sprague Dawley rats with an average body weight of 320 g.

FcA and 10D1F. FcB were administered in single doses of 4mg (-10 mg/kg), 10mg (-25 mg/kg), 40mg (-100 mg/kg) or 100mg (-250 mg/kg) by tail vein slow intravenous injection. The vector served as a negative control. Blood from 2 rats was obtained from each treatment at baseline (-24 hours), 6 hours, 24 hours, 96 hours, 168 hours and 336 hours post-dose. Antibodies in serum were quantified by ELISA.

Parameters for pharmacokinetic analysis were derived from non-compartmental models: maximum concentration (C) _max ) AUC (0-336hr), AUC (0-infinity), half-life (t) _1/2 ) Clearance (CL), dispensing quantity at steady state (V) _d )。

The results are shown in FIGS. 57A (10mg/kg), 57B (25mg/kg), 57C (100mg/kg) and 57D (250 mg/kg).

9.2 safe immunotoxicity

The toxicological effects of 10d1f. fca and 10d1f. fcb were analyzed.

Mouse

6-8 week old female BALB/c mice (20-25g) were injected intraperitoneally with a single dose of one of the following doses 10D1F. FcA and 10D1F. FcB: 200ug (. about.10 mg/kg), 500ug (. about.25 mg/kg), 2mg (. about.100 mg/kg) or 5mg (. about.250 mg/kg) or an equal volume of PBS. 3 mice were injected for each treatment and 4 mice were injected for the PBS control. Blood samples were collected 96 hours after injection and analyzed for RBC index (total RBC, hematocrit, hemoglobin, platelet count, mean corpuscular volume, mean corpuscular hemoglobin concentration) and WBC index (total WBC, total lymphocyte, neutrophil, monocyte count). Analysis was performed using a HM5 hematology analyzer.

The results are shown in fig. 58A, 58B (RBC index) and 58C (WBC index). anti-HER 3 antibodies 10d1f. fca and 10d1f. fcb had no effect on RBC index, but were found to have an effect on WBC index at higher doses (10d1f. fca 250mg/kg, 10d1f. fcb 100mg/kg, 10d1f. fcb 250 mg/kg).

Hepatotoxicity, nephrotoxicity and pancreatic toxicity were also analyzed 96 hours after injection. Fca and 10D1f fcb had no effect on the levels of alanine aminotransferase, alkaline phosphatase, albumin, total protein (liver index; fig. 58D), creatine, blood urea nitrogen, glucose or amylase (kidney and pancreas index; fig. 58E). Fca and 10d1F. fcb also did not affect the electrolyte index sodium, potassium, calcium or phosphate (fig. 58F).

Mice treated with 10d1f. fca or 10d1f. fcb showed no abnormalities after 96 hours of body weight, behavior, skin condition, oral examination, stool and urine examination, or eye examination. An increase in spleen (splenomegaly) of approximately 1.5 times the normal size was observed in mice treated with higher doses: FcA 250mg/kg 10D1F.FCB 100mg/kg,10D1F.FCB 250 mg/kg.

Further studies were performed in BALB/c mice to evaluate the toxicological effects of repeat doses of 500. mu.g (. about.25 mg/kg) of 10D1F. FcA or 10D1F. FcB. Antibodies were administered once a week for 4 weeks. Blood was obtained 28 days after the first dose. No effect of any of the antibodies on RBC, liver, kidney, pancreas or electrolyte indices was observed, no signs of clinical abnormalities were observed, and no differences were found in total nephroscopy. In mice treated with 10d1f. fca or 10d1f. fcb, a decrease in total WBC count, lymphocyte count and neutrophil count was observed, but this was considered to be non-toxic.

In another study BALB/c mice were given a single dose of 10d1f. fca or an equal volume of PBS (vector control) and analyzed after 96 hours (vector) or 336 hours (10d1f. fca). Representative results are shown in fig. 69A to 69C.

Rat

Sprague Dawley female rats (400-450g) 6-8 weeks old were injected intraperitoneally with a single dose of either 10d1F. FcA or 10d1F. FcB antibody: 4mg (10 mg/kg), 10mg (25 mg/kg), 40mg (100 mg/kg), 100mg (250 mg/kg). Blood was obtained at-24 hours, 6 hours, 24 hours, 96 hours, 168 hours, and 336 hours. Up to 366 hours after injection, there was no effect on RBC index, no toxic effect on WBC index, no effect on liver, kidney, pancreas or electrolyte index. There were no signs of clinical abnormalities, nor were differences found by gross anatomical examination (gross necropy).

Representative results obtained from rats administered 250mg/kg 10d1f. fca are shown in fig. 70A to 70C.

The absence of toxicity signals in rodent toxicology models indicates that 10D1 and variants thereof have superior clinical safety.

9.3 in vitro therapeutic efficacy analysis of cancer

The ability of anti-HER 3 antibody 10d1f. fca to inhibit tumor growth in vitro was analyzed in various tumor models: n87 cells (gastric cancer), HCC95 cells (lung cancer), FaDu cells (head and neck cancer), SNU-16 cells (gastric cancer), a549 cells (lung cancer), OvCar8 cells (ovarian cancer), ACHN cells (kidney) cancer) and HT29 cells (colorectal cancer). Fca was compared for efficacy to cetuximab (MM-121) and LJM-716 as well as other EGFR family therapies cetuximab, trastuzumab and pertuzumab of the other anti-HER 3 antibody.

Cells were treated with serial dilution concentrations of therapeutic antibody by 9-point dilution starting at 1500 ug/ml. Cell viability was measured using the CCK-8 cell proliferation assay 3-5 days after treatment. The percent cell inhibition shown is relative to cells treated with buffer only (PBS). Data points represent the average of three replicates.

The results are shown in fig. 59A to 59D. Fca showed superior tumor inhibition in various tumor models in vitro by the anti-HER 3 antibody 10D1f compared to other HER3 antibodies (59A and 59B) and EGFR family therapies (59C and 59D).

FIGS. 77A and 77B show C achieved by intraperitoneal administration of 25mg/kg of relevant antibody to mice with different anti-ErbB antibodies in vitro _max The ability of the concentration to inhibit proliferation of different cancer cell lines. Fca showed excellent ability to inhibit the growth of a variety of different cancer cell types.

9.4 in vivo therapeutic efficacy analysis of cancer

The effect of anti-HER 3 antibody clones 10d1f. fca and 10d1f. fcb on tumor growth was evaluated in an in vivo cancer model.

9.4.1A549 model

Tumor cells were injected subcutaneously into the right flank of female NCr nude mice. Antibodies (25mg/kg 10d1f. fca, 10d1f. fcb, cetuximab, LJM-716 or MM-121; each treatment n ═ 6) or vehicle (n ═ 8) were administered once every two weeks, once every six weeks.

The results are shown in FIG. 60. Both anti-HER 3 antibody clones 10d1f. fca and 10d1f. fcb showed potent efficacy in the lung cancer a549 model. Fcb was found to be particularly effective and regressed.

9.4.2FaDu model

Tumor cells were inserted subcutaneously into the right flank of female NCr nude mice with matrigel. Antibodies (10 and 25mg/kg 10d1f. fca and 10d1f. fcb, or 25mg/kg cetuximab, trastuzumab, pertuzumab, LJM-716 or MM-121; n ═ 6 for each treatment method) or vehicle (n ═ 6) once a week for six weeks.

The results are shown in FIG. 61. In the FaDu model of head and neck cancer, anti-HER 3 antibody clones 10d1f. fca and 10d1f. fcb were both found to be effective in preventing tumor growth.

9.4.3OvCar8 model

Tumor cells were inserted subcutaneously into the right flank of female NCr nude mice with matrigel. Antibodies (10 and 25mg/kg 10d1f. fca, or 25mg/kg cetuximab, LJM-716 or MM-121; n ═ 6) or vehicle (n ═ 6) were administered once a week for six consecutive weeks.

The results are shown in FIG. 62. Fca was found to be effective in reducing tumor volume at high doses with the anti-HER 3 antibody clone 10d1f.

9.4.4N87 model

Tumor cells were inserted subcutaneously into the right flank of female NCr nude mice with matrigel. Antibodies (25mg/kg 10d1f. fca or 50mg/kg trastuzumab, LJM-716 or MM-121; n ═ 6 for each treatment method) or vehicle (n ═ 6) were administered once every two weeks, once every six weeks.

The results are shown in FIG. 74. Fca was found to be effective in preventing tumor growth in the gastric cancer N87 model by the anti-HER 3 antibody 10d1f.

Example 10: analysis for inhibiting proliferation of BRAFV600E mutant thyroid cancer cell line

The following cell lines were studied:

cell lines	Cancer type	Mutations
			SW1736	Anaplastic thyroid cancer	BRAF V600E
BHT101	Anaplastic thyroid cancer	BRAF V600E
			BCPAP	Papillary carcinoma of thyroid	BRAF V600E and p53 mutations

Surface expression of EGFR family members in cells was studied by flow cytometry. Briefly, 300,000 cells were incubated with 20 μ g/ml of 10d1f. fca, cetuximab, or trastuzumab at 4 ℃ for 1 hour. As a secondary antibody, Alexafluor 488-conjugated anti-human antibody at a concentration of 10. mu.g/ml was used (40 minutes at 4 ℃).

The results are shown in fig. 66A to 66C. SW1736, BHT101 and BCPAP cells were shown to express EGFR, HER2 and HER 3.

The inventors investigated the ability of different HER3 binding antibodies to inhibit the in vitro proliferation of different thyroid cancer cell lines bearing the V600E BRAF mutation.

Briefly, cells of different cell lines were plated at 1.5X 10 ⁵ Cells/well were seeded at density and the next day the cells were treated with 10-point serial dilutions starting from 1000 μ g/ml with 10d1f. fca, selitumumab, LJM-716, pertuzumab or isotype control antibody. After 3 days, proliferation was measured using the CCK-8 cell proliferation assay. Percent proliferation inhibition was calculated relative to cells treated with an equal volume of PBS instead of antibody.

The results are shown in FIGS. 67A to 67C. Fca was found to inhibit proliferation of cell lines carrying the BRAF V600E mutation more effectively than any other anti-HER 3 antibody analyzed.

In a further experiment, the ability of a combination of 10d1f. fca and vemurafenib (vemurafenib) to inhibit the in vitro proliferation of different thyroid cancer cell lines with the V600E BRAF mutation was investigated.

Cells were plated at 1.5X 10 ⁵ Cells/well density were seeded and on the following day, cells were treated with 10-point serial dilutions of 10d1f. fca or isotype control antibody starting from 1000 μ g/ml in the presence or absence of 200nM vemurafenib. After 3 days, CCK was used-8 cell proliferation assay to determine proliferation. Percent proliferation inhibition was calculated relative to cells treated with an equal volume of PBS instead of antibody.

The results are shown in FIGS. 68A to 68C. Fca was found to enhance the ability of vemurafenib to inhibit the proliferation of SW1736 and BHT101 cells, which SW1736 and BHT101 cells are susceptible to vemurafenib. Fca was also found to be a potent inhibitor against vemurafenib BCPAP cell proliferation.

Example 11: in vivo assay for inhibition of HER 3-mediated signaling

Fca was investigated for its ability to inhibit HER 3-mediated signaling in vivo.

Will be 1 × 10 ⁶ FaDu or OvCar8 cells were injected subcutaneously into NCr nude mice to establish ectopic xenograft tumors.

Once the tumor volume exceeds 100mm ³ Mice were treated with an intraperitoneal injection of 10d1f. fca or an equal volume of vehicle (control) at a dose of 25mg/kg every two weeks. After 4 weeks, tumors were harvested. Protein extracts were prepared from tumors and quantified by the Bradford assay, 50 μ g samples were separated by SDS-PAGE and analyzed by western blot using antibodies to determine in vivo phosphorylation of HER3 and AKT as described in example 4.3.

The results are shown in FIG. 71. Fca was found to inhibit phosphorylation of HER3 and AKT in tumor cells in vivo.

Example 12: internalization assay for anti-HER 3 antibodies

The inventors investigated the internalization of HER3 expressing cells against the anti-HER 3 antibody.

Briefly, 100,000 HEK293 cells engineered to express HER3, HCC95, N87, or OVCAR8 cells were seeded into wells of 96-well tissue culture plates and incubated at 37 ℃, 5% CO ₂ Cultured overnight in the medium. The cells were then treated with 120nM 10D1F. FcA, LJM-716, selitumumab or trastuzumab and 360nM of pHrodo iFL green reagent at 37 ℃ with 5% CO ₂ And (4) carrying out incubation. Cultured cells were imaged in 4 different fields of view per well for 24 hours every 30 minutes. Quantification of FITC-N at each field over 24 hours Maximum signal strength in the track.

The results are shown in FIG. 72. Moderate internalization of LJM-716 and selitumab was observed in OvCar8 cells, whereas moderate internalization of trastuzumab was observed in N87 cells.

No significant internalization of 10d1f. fca was observed in HCC95, N87, and OvCar8 cells.

As expected, significant internalization of 10d1f. fca, LJM-716 and selitumumab (seribanumab) was observed in HEK293 cells overexpressing HER 3.

In further experiments, antibody internalization was studied by flow cytometry.

N87 cells were seeded at a density of 50,000 cells/well in wells of 96-well tissue culture plates and allowed to adhere overnight (37 ℃, 5% CO) ₂ ). Fca or trastuzumab, 10d1f. is mixed with the labeling reagent and the labeled complex is added to the cells. Samples were collected at time points of 0 min, 10 min, 30 min, 1 hr, 2 hr, and 4.5 hr by aspirating the cell culture medium, washing with PBS, and treating with Accutase. The activity of Accutase was neutralized and the cells were resuspended in FACs buffer and analyzed by flow cytometry.

The results are shown in FIGS. 73A and 73B. Minimal internalization of fca. In contrast, substantial internalization of trastuzumab, the anti-HER 2 antibody, was observed.

Example 13: use of HER3 binding antibodies in immunohistochemistry

The ability of the anti-HER 3 antibody 10D1F in mIgG2a form to be used in immunohistochemistry to detect human HER3 protein was evaluated.

The sections were processed using Bond reagents (Leica Biosystems). Commercially available arrays of frozen tissue sections were obtained. The slides were dried in a desiccator for 10 minutes, then treated as follows, and water washed and/or TBS-T rinsed between steps: (i) fixation was performed by treatment with 100% acetone for 10 min at room temperature; (ii) by reaction with 3% (v/v) H at room temperature ₂ O ₂ Treatment for 15 minutes to block endogenous peroxidase; (iii) sealing with 10% goat serum for 30 min at room temperature(iv) 1: incubation with 10D1F-mIgG2a at 250 dilution overnight at 4 ℃, (v) goat anti-mouse antibody conjugated with HRP incubate-polymer conjugate was left for 30 minutes at room temperature, (vi) developed with bond Mixed DAB reference for 5 minutes at room temperature, then washed with deionized water and 1x bond eluent to stop the reaction.

The slides were then dehydrated, fixed in synthetic fixing media, and subjected to high resolution scanning.

The results are shown in FIGS. 75A and 75B. 10D1F preferentially stained sections of malignant human tissue with low cross-reactivity with normal tissue.

In further experiments, a549 xenografts were harvested in cold PBS, embedded in OCT cryo-embedding medium, frozen in dry ice and stored at-80 ℃.10 μm sections were obtained using a cryostat.

The slides were dried in a desiccator for 10 minutes, then treated as follows, with water washes and/or TBS-T rinses between steps: (i) fixation was performed by treatment with 100% acetone for 10 min at room temperature; (ii) by reaction with 3% (v/v) H at room temperature ₂ O ₂ Treatment for 15 minutes to block endogenous peroxidase; (iii) blocking by treatment with 10% goat serum for 30 min at room temperature; (iv) incubation with 10d1f. fca diluted 1:50 to 8.8mg/ml solution, or with 1: rabbit anti-HER 3 (cat No. 10201-T24) from Sino organisms at 200 dilution was incubated overnight, (v) incubated with invitrogen's F (ab') 2-goat anti-human IgG (H + L) HRP (a24470) (1: 500) or HRP-polymer conjugated goat anti-rabbit antibody for 30 minutes at room temperature, (vi) incubated with Bond Mixed DAB Refine for 5 minutes at room temperature, then washed with deionized water and 1x Wash (Bond Wash) to stop the reaction.

The slides were then counterstained with hematoxylin, dehydrated, fixed in synthetic fixing media, and scanned at high resolution.

The results are shown in FIG. 76. Fca showed specific membrane and cytoplasmic staining of a549 tumor xenograft frozen sections.

Example 14: HER3 binding antibody in patient-derived ovarian cancer xenograft model containing CLU-NRG1 fusion The therapeutic efficacy of

The inventors investigated the therapeutic efficacy of 10D1F in a patient-derived ovarian cancer xenograft model comprising a CLU-NRG1 fusion.

Female BALB/c nude mice, approximately 5-7 weeks old, were placed under specific pathogen-free conditions and treated according to Institutional Animal Care and Use Committee (IACUC) guidelines.

The ovarian cancer model comprising the CLU-NRG1 fusion was established by subcutaneous inoculation of the right side of mice with four patient-derived xenografts OV6308 (imperial biosciences). OV6308 is an ovarian high-grade serous adenocarcinoma from a 51 year old female patient comprising the CLU-NRG1 gene fusion.

Tumor volumes were measured 3 times per week using digital calipers and calculated using the formula [ L × W2/2 ]. Once the tumor length of the control arm >1.5cm, the study endpoint was considered to have been reached.

Mice were administered intraperitoneally every two weeks with either (i)25mg/kg 10d1f. fca (i.e. example 2.2[16]) or (i)25mg/kg hIgG1 isotype control antibody (n ═ 10 per treatment group).

The results are shown in FIG. 79. Fca treatment was found to be extremely effective, inhibiting tumor growth by 111% relative to isotype control treated groups.

Sequence listing

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Cys Pro Pro Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys

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Glu Pro Cys Gly Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser

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His Glu Ala Glu Cys Phe Ser Cys His Pro Glu Cys Gln Pro Met Glu

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Val Leu Gly Ala Lys Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln Asn

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Ala Phe Gln Gly Pro Gly His Gln Ala Pro His Val His Tyr Ala

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Arg Leu Lys Thr Leu Arg Ser Leu Glu Ala Thr Asp Ser Ala Phe

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Glu Pro Asn Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala

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Ser Cys Pro His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala

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Cys Pro Pro Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys

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Asn Thr Asn Ser Ser His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu

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Thr Glu Ile Leu Ser Gly Gly Val Tyr Ile Glu Lys Asn Asp Lys Leu

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Cys His Met Asp Thr Ile Asp Trp Arg Asp Ile Val Arg Asp Arg Asp

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Ala Glu Ile Val Val Lys Asp Asn Gly Arg Ser Cys Pro Pro Cys His

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Thr Leu Thr Lys Thr Ile Cys Ala Pro Gln Cys Asn Gly His Cys Phe

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Gly Pro Asn Pro Asn Gln Cys Cys His Asp Glu Cys Ala Gly Gly Cys

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Leu Thr Phe Gln Leu Glu Pro Asn Pro His Thr Lys Tyr Gln Tyr Gly

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Ser Cys Val Arg Ala Cys Pro Pro Asp Lys Met Glu Val Asp Lys Asn

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Gly Leu Lys Met Cys Glu Pro Cys Gly Gly Leu Cys Pro Lys Ala Cys

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Glu Gly Thr Gly Ser Gly Ser Arg Phe Gln Thr Val Asp Ser Ser Asn

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Ile Asp Gly Phe Val Asn Cys Thr Lys Ile Leu Gly Asn Leu Asp Phe

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Leu Ile Thr Gly Leu Asn Gly Asp Pro Trp His Lys Ile Pro Ala Leu

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Asp Pro Glu Lys Leu Asn Val Phe Arg Thr Val Arg Glu Ile Thr Gly

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Tyr Leu Asn Ile Gln Ser Trp Pro Pro His Met His Asn Phe Ser Val

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Phe Ser Asn Leu Thr Thr Ile Gly Gly Arg Ser Leu Tyr Asn Arg Gly

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Phe Ser Leu Leu Ile Met Lys Asn Leu Asn Val Thr Ser Leu Gly Phe

370 375 380

Arg Ser Leu Lys Glu Ile Ser Ala Gly Arg Ile Tyr Ile Ser Ala Asn

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Arg Gln Leu Cys Tyr His His Ser Leu Asn Trp Thr Lys Val Leu Arg

405 410 415

Gly Pro Thr Glu Glu Arg Leu Asp Ile Lys His Asn Arg Pro Arg Arg

420 425 430

Asp Cys Val Ala Glu Gly Lys Val Cys Asp Pro Leu Cys Ser Ser Gly

435 440 445

Gly Cys Trp Gly Pro Gly Pro Gly Gln Cys Leu Ser Cys Arg Asn Tyr

450 455 460

Ser Arg Gly Gly Val Cys Val Thr His Cys Asn Phe Leu Asn Gly Glu

465 470 475 480

Pro Arg Glu Phe Ala His Glu Ala Glu Cys Phe Ser Cys His Pro Glu

485 490 495

Cys Gln Pro Met Glu Gly Thr Ala Thr Cys Asn Gly Ser Gly Ser Asp

500 505 510

Thr Cys Ala Gln Cys Ala His Phe Arg Asp Gly Pro His Cys Val Ser

515 520 525

Ser Cys Pro His Gly Val Leu Gly Ala Lys Gly Pro Ile Tyr Lys Tyr

530 535 540

Pro Asp Val Gln Asn Glu Cys Arg Pro Cys His Glu Asn Cys Thr Gln

545 550 555 560

Gly Cys Lys Gly Pro Glu Leu Gln Asp Cys Leu Gly Gln Thr Leu Val

565 570 575

Leu Ile Gly Lys Thr His Leu Thr Met Ala Leu Thr Val Ile Ala Gly

580 585 590

Leu Val Val Ile Phe Met Met Leu Gly Gly Thr Phe Leu Tyr Trp Arg

595 600 605

Gly Arg Arg Ile Gln Asn Lys Arg Ala Met Arg Arg Tyr Leu Glu Arg

610 615 620

Gly Glu Ser Ile Glu Pro Leu Asp Pro Ser Glu Lys Ala Asn Lys Val

625 630 635 640

Leu Ala Arg Ile Phe Lys Glu Thr Glu Leu Arg Lys Leu Lys Val Leu

645 650 655

Gly Ser Gly Val Phe Gly Thr Val His Lys Gly Val Trp Ile Pro Glu

660 665 670

Gly Glu Ser Ile Lys Ile Pro Val Cys Ile Lys Val Ile Glu Asp Lys

675 680 685

Ser Gly Arg Gln Ser Phe Gln Ala Val Thr Asp His Met Leu Ala Ile

690 695 700

Gly Ser Leu Asp His Ala His Ile Val Arg Leu Leu Gly Leu Cys Pro

705 710 715 720

Gly Ser Ser Leu Gln Leu Val Thr Gln Tyr Leu Pro Leu Gly Ser Leu

725 730 735

Leu Asp His Val Arg Gln His Arg Gly Ala Leu Gly Pro Gln Leu Leu

740 745 750

Leu Asn Trp Gly Val Gln Ile Ala Lys Gly Met Tyr Tyr Leu Glu Glu

755 760 765

His Gly Met Val His Arg Asn Leu Ala Ala Arg Asn Val Leu Leu Lys

770 775 780

Ser Pro Ser Gln Val Gln Val Ala Asp Phe Gly Val Ala Asp Leu Leu

785 790 795 800

Pro Pro Asp Asp Lys Gln Leu Leu Tyr Ser Glu Ala Lys Thr Pro Ile

805 810 815

Lys Trp Met Ala Leu Glu Ser Ile His Phe Gly Lys Tyr Thr His Gln

820 825 830

Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe

835 840 845

Gly Ala Glu Pro Tyr Ala Gly Leu Arg Leu Ala Glu Val Pro Asp Leu

850 855 860

Leu Glu Lys Gly Glu Arg Leu Ala Gln Pro Gln Ile Cys Thr Ile Asp

865 870 875 880

Val Tyr Met Val Met Val Lys Cys Trp Met Ile Asp Glu Asn Ile Arg

885 890 895

Pro Thr Phe Lys Glu Leu Ala Asn Glu Phe Thr Arg Met Ala Arg Asp

900 905 910

Pro Pro Arg Tyr Leu Val Ile Lys Arg Glu Ser Gly Pro Gly Ile Ala

915 920 925

Pro Gly Pro Glu Pro His Gly Leu Thr Asn Lys Lys Leu Glu Glu Val

930 935 940

Glu Leu Glu Pro Glu Leu Asp Leu Asp Leu Asp Leu Glu Ala Glu Glu

945 950 955 960

Asp Asn Leu Ala Thr Thr Thr Leu Gly Ser Ala Leu Ser Leu Pro Val

965 970 975

Gly Thr Leu Asn Arg Pro Arg Gly Ser Gln Ser Leu Leu Ser Pro Ser

980 985 990

Ser Gly Tyr Met Pro Met Asn Gln Gly Asn Leu Gly Glu Ser Cys Gln

995 1000 1005

Glu Ser Ala Val Ser Gly Ser Ser Glu Arg Cys Pro Arg Pro Val

1010 1015 1020

Ser Leu His Pro Met Pro Arg Gly Cys Leu Ala Ser Glu Ser Ser

1025 1030 1035

Glu Gly His Val Thr Gly Ser Glu Ala Glu Leu Gln Glu Lys Val

1040 1045 1050

Ser Met Cys Arg Ser Arg Ser Arg Ser Arg Ser Pro Arg Pro Arg

1055 1060 1065

Gly Asp Ser Ala Tyr His Ser Gln Arg His Ser Leu Leu Thr Pro

1070 1075 1080

Val Thr Pro Leu Ser Pro Pro Gly Leu Glu Glu Glu Asp Val Asn

1085 1090 1095

Gly Tyr Val Met Pro Asp Thr His Leu Lys Gly Thr Pro Ser Ser

1100 1105 1110

Arg Glu Gly Thr Leu Ser Ser Val Gly Leu Ser Ser Val Leu Gly

1115 1120 1125

Thr Glu Glu Glu Asp Glu Asp Glu Glu Tyr Glu Tyr Met Asn Arg

1130 1135 1140

Arg Arg Arg His Ser Pro Pro His Pro Pro Arg Pro Ser Ser Leu

1145 1150 1155

Glu Glu Leu Gly Tyr Glu Tyr Met Asp Val Gly Ser Asp Leu Ser

1160 1165 1170

Ala Ser Leu Gly Ser Thr Gln Ser Cys Pro Leu His Pro Val Pro

1175 1180 1185

Ile Met Pro Thr Ala Gly Thr Thr Pro Asp Glu Asp Tyr Glu Tyr

1190 1195 1200

Met Asn Arg Gln Arg Asp Gly Gly Gly Pro Gly Gly Asp Tyr Ala

1205 1210 1215

Ala Met Gly Ala Cys Pro Ala Ser Glu Gln Gly Tyr Glu Glu Met

1220 1225 1230

Arg Ala Phe Gln Gly Pro Gly His Gln Ala Pro His Val His Tyr

1235 1240 1245

Ala Arg Leu Lys Thr Leu Arg Ser Leu Glu Ala Thr Asp Ser Ala

1250 1255 1260

Phe Asp Asn Pro Asp Tyr Trp His Ser Arg Leu Phe Pro Lys Ala

1265 1270 1275

Asn Ala Gln Arg Thr

1280

<210> 5

<211> 699

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 5

Met Ala Leu Thr Val Ile Ala Gly Leu Val Val Ile Phe Met Met Leu

1 5 10 15

Gly Gly Thr Phe Leu Tyr Trp Arg Gly Arg Arg Ile Gln Asn Lys Arg

20 25 30

Ala Met Arg Arg Tyr Leu Glu Arg Gly Glu Ser Ile Glu Pro Leu Asp

35 40 45

Pro Ser Glu Lys Ala Asn Lys Val Leu Ala Arg Ile Phe Lys Glu Thr

50 55 60

Glu Leu Arg Lys Leu Lys Val Leu Gly Ser Gly Val Phe Gly Thr Val

65 70 75 80

His Lys Gly Val Trp Ile Pro Glu Gly Glu Ser Ile Lys Ile Pro Val

85 90 95

Cys Ile Lys Val Ile Glu Asp Lys Ser Gly Arg Gln Ser Phe Gln Ala

100 105 110

Val Thr Asp His Met Leu Ala Ile Gly Ser Leu Asp His Ala His Ile

115 120 125

Val Arg Leu Leu Gly Leu Cys Pro Gly Ser Ser Leu Gln Leu Val Thr

130 135 140

Gln Tyr Leu Pro Leu Gly Ser Leu Leu Asp His Val Arg Gln His Arg

145 150 155 160

Gly Ala Leu Gly Pro Gln Leu Leu Leu Asn Trp Gly Val Gln Ile Ala

165 170 175

Lys Gly Met Tyr Tyr Leu Glu Glu His Gly Met Val His Arg Asn Leu

180 185 190

Ala Ala Arg Asn Val Leu Leu Lys Ser Pro Ser Gln Val Gln Val Ala

195 200 205

Asp Phe Gly Val Ala Asp Leu Leu Pro Pro Asp Asp Lys Gln Leu Leu

210 215 220

Tyr Ser Glu Ala Lys Thr Pro Ile Lys Trp Met Ala Leu Glu Ser Ile

225 230 235 240

His Phe Gly Lys Tyr Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val

245 250 255

Thr Val Trp Glu Leu Met Thr Phe Gly Ala Glu Pro Tyr Ala Gly Leu

260 265 270

Arg Leu Ala Glu Val Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Ala

275 280 285

Gln Pro Gln Ile Cys Thr Ile Asp Val Tyr Met Val Met Val Lys Cys

290 295 300

Trp Met Ile Asp Glu Asn Ile Arg Pro Thr Phe Lys Glu Leu Ala Asn

305 310 315 320

Glu Phe Thr Arg Met Ala Arg Asp Pro Pro Arg Tyr Leu Val Ile Lys

325 330 335

Arg Glu Ser Gly Pro Gly Ile Ala Pro Gly Pro Glu Pro His Gly Leu

340 345 350

Thr Asn Lys Lys Leu Glu Glu Val Glu Leu Glu Pro Glu Leu Asp Leu

355 360 365

Asp Leu Asp Leu Glu Ala Glu Glu Asp Asn Leu Ala Thr Thr Thr Leu

370 375 380

Gly Ser Ala Leu Ser Leu Pro Val Gly Thr Leu Asn Arg Pro Arg Gly

385 390 395 400

Ser Gln Ser Leu Leu Ser Pro Ser Ser Gly Tyr Met Pro Met Asn Gln

405 410 415

Gly Asn Leu Gly Glu Ser Cys Gln Glu Ser Ala Val Ser Gly Ser Ser

420 425 430

Glu Arg Cys Pro Arg Pro Val Ser Leu His Pro Met Pro Arg Gly Cys

435 440 445

Leu Ala Ser Glu Ser Ser Glu Gly His Val Thr Gly Ser Glu Ala Glu

450 455 460

Leu Gln Glu Lys Val Ser Met Cys Arg Ser Arg Ser Arg Ser Arg Ser

465 470 475 480

Pro Arg Pro Arg Gly Asp Ser Ala Tyr His Ser Gln Arg His Ser Leu

485 490 495

Leu Thr Pro Val Thr Pro Leu Ser Pro Pro Gly Leu Glu Glu Glu Asp

500 505 510

Val Asn Gly Tyr Val Met Pro Asp Thr His Leu Lys Gly Thr Pro Ser

515 520 525

Ser Arg Glu Gly Thr Leu Ser Ser Val Gly Leu Ser Ser Val Leu Gly

530 535 540

Thr Glu Glu Glu Asp Glu Asp Glu Glu Tyr Glu Tyr Met Asn Arg Arg

545 550 555 560

Arg Arg His Ser Pro Pro His Pro Pro Arg Pro Ser Ser Leu Glu Glu

565 570 575

Leu Gly Tyr Glu Tyr Met Asp Val Gly Ser Asp Leu Ser Ala Ser Leu

580 585 590

Gly Ser Thr Gln Ser Cys Pro Leu His Pro Val Pro Ile Met Pro Thr

595 600 605

Ala Gly Thr Thr Pro Asp Glu Asp Tyr Glu Tyr Met Asn Arg Gln Arg

610 615 620

Asp Gly Gly Gly Pro Gly Gly Asp Tyr Ala Ala Met Gly Ala Cys Pro

625 630 635 640

Ala Ser Glu Gln Gly Tyr Glu Glu Met Arg Ala Phe Gln Gly Pro Gly

645 650 655

His Gln Ala Pro His Val His Tyr Ala Arg Leu Lys Thr Leu Arg Ser

660 665 670

Leu Glu Ala Thr Asp Ser Ala Phe Asp Asn Pro Asp Tyr Trp His Ser

675 680 685

Arg Leu Phe Pro Lys Ala Asn Ala Gln Arg Thr

690 695

<210> 6

<211> 1323

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 6

Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr Leu Asn Gly

1 5 10 15

Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr Leu Tyr Lys

20 25 30

Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu Ile Val Leu

35 40 45

Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile Arg Glu Val

50 55 60

Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr Leu Pro Leu

65 70 75 80

Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp Gly Lys Phe

85 90 95

Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser His Ala Leu

100 105 110

Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser Gly Gly Val

115 120 125

Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr Ile Asp Trp

130 135 140

Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val Lys Asp Asn

145 150 155 160

Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly Arg Cys Trp

165 170 175

Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr Ile Cys Ala

180 185 190

Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn Gln Cys Cys

195 200 205

His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp Thr Asp Cys

210 215 220

Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val Pro Arg Cys

225 230 235 240

Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu Glu Pro Asn

245 250 255

Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala Ser Cys Pro

260 265 270

His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala Cys Pro Pro

275 280 285

Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys Glu Pro Cys

290 295 300

Gly Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser Gly Ser Arg

305 310 315 320

Phe Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val Asn Cys Thr

325 330 335

Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu Asn Gly Asp

340 345 350

Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu Asn Val Phe

355 360 365

Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gln Ser Trp Pro

370 375 380

Pro His Met His Asn Phe Ser Val Phe Ser Asn Leu Thr Thr Ile Gly

385 390 395 400

Gly Arg Ser Leu Tyr Asn Arg Gly Phe Ser Leu Leu Ile Met Lys Asn

405 410 415

Leu Asn Val Thr Ser Leu Gly Phe Arg Ser Leu Lys Glu Ile Ser Ala

420 425 430

Gly Arg Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr His His Ser

435 440 445

Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr Glu Glu Arg Leu Asp

450 455 460

Ile Lys His Asn Arg Pro Arg Arg Asp Cys Val Ala Glu Gly Lys Val

465 470 475 480

Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys Trp Gly Pro Gly Pro Gly

485 490 495

Gln Cys Leu Ser Cys Arg Asn Tyr Ser Arg Gly Gly Val Cys Val Thr

500 505 510

His Cys Asn Phe Leu Asn Gly Glu Pro Arg Glu Phe Ala His Glu Ala

515 520 525

Glu Cys Phe Ser Cys His Pro Glu Cys Gln Pro Met Glu Gly Thr Ala

530 535 540

Thr Cys Asn Gly Ser Gly Ser Asp Thr Cys Ala Gln Cys Ala His Phe

545 550 555 560

Arg Asp Gly Pro His Cys Val Ser Ser Cys Pro His Gly Val Leu Gly

565 570 575

Ala Lys Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln Asn Glu Cys Arg

580 585 590

Pro Cys His Glu Asn Cys Thr Gln Gly Cys Lys Gly Pro Glu Leu Gln

595 600 605

Asp Cys Leu Gly Gln Thr Leu Val Leu Ile Gly Lys Thr His Leu Thr

610 615 620

Met Ala Leu Thr Val Ile Ala Gly Leu Val Val Ile Phe Met Met Leu

625 630 635 640

Gly Gly Thr Phe Leu Tyr Trp Arg Gly Arg Arg Ile Gln Asn Lys Arg

645 650 655

Ala Met Arg Arg Tyr Leu Glu Arg Gly Glu Ser Ile Glu Pro Leu Asp

660 665 670

Pro Ser Glu Lys Ala Asn Lys Val Leu Ala Arg Ile Phe Lys Glu Thr

675 680 685

Glu Leu Arg Lys Leu Lys Val Leu Gly Ser Gly Val Phe Gly Thr Val

690 695 700

His Lys Gly Val Trp Ile Pro Glu Gly Glu Ser Ile Lys Ile Pro Val

705 710 715 720

Cys Ile Lys Val Ile Glu Asp Lys Ser Gly Arg Gln Ser Phe Gln Ala

725 730 735

Val Thr Asp His Met Leu Ala Ile Gly Ser Leu Asp His Ala His Ile

740 745 750

Val Arg Leu Leu Gly Leu Cys Pro Gly Ser Ser Leu Gln Leu Val Thr

755 760 765

Gln Tyr Leu Pro Leu Gly Ser Leu Leu Asp His Val Arg Gln His Arg

770 775 780

Gly Ala Leu Gly Pro Gln Leu Leu Leu Asn Trp Gly Val Gln Ile Ala

785 790 795 800

Lys Gly Met Tyr Tyr Leu Glu Glu His Gly Met Val His Arg Asn Leu

805 810 815

Ala Ala Arg Asn Val Leu Leu Lys Ser Pro Ser Gln Val Gln Val Ala

820 825 830

Asp Phe Gly Val Ala Asp Leu Leu Pro Pro Asp Asp Lys Gln Leu Leu

835 840 845

Tyr Ser Glu Ala Lys Thr Pro Ile Lys Trp Met Ala Leu Glu Ser Ile

850 855 860

His Phe Gly Lys Tyr Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val

865 870 875 880

Thr Val Trp Glu Leu Met Thr Phe Gly Ala Glu Pro Tyr Ala Gly Leu

885 890 895

Arg Leu Ala Glu Val Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Ala

900 905 910

Gln Pro Gln Ile Cys Thr Ile Asp Val Tyr Met Val Met Val Lys Cys

915 920 925

Trp Met Ile Asp Glu Asn Ile Arg Pro Thr Phe Lys Glu Leu Ala Asn

930 935 940

Glu Phe Thr Arg Met Ala Arg Asp Pro Pro Arg Tyr Leu Val Ile Lys

945 950 955 960

Arg Glu Ser Gly Pro Gly Ile Ala Pro Gly Pro Glu Pro His Gly Leu

965 970 975

Thr Asn Lys Lys Leu Glu Glu Val Glu Leu Glu Pro Glu Leu Asp Leu

980 985 990

Asp Leu Asp Leu Glu Ala Glu Glu Asp Asn Leu Ala Thr Thr Thr Leu

995 1000 1005

Gly Ser Ala Leu Ser Leu Pro Val Gly Thr Leu Asn Arg Pro Arg

1010 1015 1020

Gly Ser Gln Ser Leu Leu Ser Pro Ser Ser Gly Tyr Met Pro Met

1025 1030 1035

Asn Gln Gly Asn Leu Gly Glu Ser Cys Gln Glu Ser Ala Val Ser

1040 1045 1050

Gly Ser Ser Glu Arg Cys Pro Arg Pro Val Ser Leu His Pro Met

1055 1060 1065

Pro Arg Gly Cys Leu Ala Ser Glu Ser Ser Glu Gly His Val Thr

1070 1075 1080

Gly Ser Glu Ala Glu Leu Gln Glu Lys Val Ser Met Cys Arg Ser

1085 1090 1095

Arg Ser Arg Ser Arg Ser Pro Arg Pro Arg Gly Asp Ser Ala Tyr

1100 1105 1110

His Ser Gln Arg His Ser Leu Leu Thr Pro Val Thr Pro Leu Ser

1115 1120 1125

Pro Pro Gly Leu Glu Glu Glu Asp Val Asn Gly Tyr Val Met Pro

1130 1135 1140

Asp Thr His Leu Lys Gly Thr Pro Ser Ser Arg Glu Gly Thr Leu

1145 1150 1155

Ser Ser Val Gly Leu Ser Ser Val Leu Gly Thr Glu Glu Glu Asp

1160 1165 1170

Glu Asp Glu Glu Tyr Glu Tyr Met Asn Arg Arg Arg Arg His Ser

1175 1180 1185

Pro Pro His Pro Pro Arg Pro Ser Ser Leu Glu Glu Leu Gly Tyr

1190 1195 1200

Glu Tyr Met Asp Val Gly Ser Asp Leu Ser Ala Ser Leu Gly Ser

1205 1210 1215

Thr Gln Ser Cys Pro Leu His Pro Val Pro Ile Met Pro Thr Ala

1220 1225 1230

Gly Thr Thr Pro Asp Glu Asp Tyr Glu Tyr Met Asn Arg Gln Arg

1235 1240 1245

Asp Gly Gly Gly Pro Gly Gly Asp Tyr Ala Ala Met Gly Ala Cys

1250 1255 1260

Pro Ala Ser Glu Gln Gly Tyr Glu Glu Met Arg Ala Phe Gln Gly

1265 1270 1275

Pro Gly His Gln Ala Pro His Val His Tyr Ala Arg Leu Lys Thr

1280 1285 1290

Leu Arg Ser Leu Glu Ala Thr Asp Ser Ala Phe Asp Asn Pro Asp

1295 1300 1305

Tyr Trp His Ser Arg Leu Phe Pro Lys Ala Asn Ala Gln Arg Thr

1310 1315 1320

<210> 7

<211> 164

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 7

Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr Leu Asn Gly

1 5 10 15

Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr Leu Tyr Lys

20 25 30

Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu Ile Val Leu

35 40 45

Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile Arg Glu Val

50 55 60

Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr Leu Pro Leu

65 70 75 80

Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp Gly Lys Phe

85 90 95

Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser His Ala Leu

100 105 110

Arg Gln Leu Arg Leu Thr Gln Leu Thr Gly Gln Phe Pro Met Val Pro

115 120 125

Ser Gly Leu Thr Pro Gln Pro Ala Gln Asp Trp Tyr Leu Leu Asp Asp

130 135 140

Asp Pro Arg Leu Leu Thr Leu Ser Ala Ser Ser Lys Val Pro Val Thr

145 150 155 160

Leu Ala Ala Val

<210> 8

<211> 312

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 8

Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr Leu Asn Gly

1 5 10 15

Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr Leu Tyr Lys

20 25 30

Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu Ile Val Leu

35 40 45

Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile Arg Glu Val

50 55 60

Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr Leu Pro Leu

65 70 75 80

Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp Gly Lys Phe

85 90 95

Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser His Ala Leu

100 105 110

Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser Gly Gly Val

115 120 125

Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr Ile Asp Trp

130 135 140

Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val Lys Asp Asn

145 150 155 160

Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly Arg Cys Trp

165 170 175

Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr Ile Cys Ala

180 185 190

Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn Gln Cys Cys

195 200 205

His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp Thr Asp Cys

210 215 220

Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val Pro Arg Cys

225 230 235 240

Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu Glu Pro Asn

245 250 255

Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala Ser Cys Pro

260 265 270

His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala Cys Pro Pro

275 280 285

Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys Glu Pro Cys

290 295 300

Gly Gly Leu Cys Pro Lys Ala Phe

305 310

<210> 9

<211> 624

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 9

Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr Leu Asn Gly

1 5 10 15

Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr Leu Tyr Lys

20 25 30

Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu Ile Val Leu

35 40 45

Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile Arg Glu Val

50 55 60

Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr Leu Pro Leu

65 70 75 80

Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp Gly Lys Phe

85 90 95

Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser His Ala Leu

100 105 110

Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser Gly Gly Val

115 120 125

Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr Ile Asp Trp

130 135 140

Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val Lys Asp Asn

145 150 155 160

Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly Arg Cys Trp

165 170 175

Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr Ile Cys Ala

180 185 190

Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn Gln Cys Cys

195 200 205

His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp Thr Asp Cys

210 215 220

Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val Pro Arg Cys

225 230 235 240

Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu Glu Pro Asn

245 250 255

Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala Ser Cys Pro

260 265 270

His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala Cys Pro Pro

275 280 285

Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys Glu Pro Cys

290 295 300

Gly Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser Gly Ser Arg

305 310 315 320

Phe Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val Asn Cys Thr

325 330 335

Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu Asn Gly Asp

340 345 350

Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu Asn Val Phe

355 360 365

Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gln Ser Trp Pro

370 375 380

Pro His Met His Asn Phe Ser Val Phe Ser Asn Leu Thr Thr Ile Gly

385 390 395 400

Gly Arg Ser Leu Tyr Asn Arg Gly Phe Ser Leu Leu Ile Met Lys Asn

405 410 415

Leu Asn Val Thr Ser Leu Gly Phe Arg Ser Leu Lys Glu Ile Ser Ala

420 425 430

Gly Arg Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr His His Ser

435 440 445

Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr Glu Glu Arg Leu Asp

450 455 460

Ile Lys His Asn Arg Pro Arg Arg Asp Cys Val Ala Glu Gly Lys Val

465 470 475 480

Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys Trp Gly Pro Gly Pro Gly

485 490 495

Gln Cys Leu Ser Cys Arg Asn Tyr Ser Arg Gly Gly Val Cys Val Thr

500 505 510

His Cys Asn Phe Leu Asn Gly Glu Pro Arg Glu Phe Ala His Glu Ala

515 520 525

Glu Cys Phe Ser Cys His Pro Glu Cys Gln Pro Met Glu Gly Thr Ala

530 535 540

Thr Cys Asn Gly Ser Gly Ser Asp Thr Cys Ala Gln Cys Ala His Phe

545 550 555 560

Arg Asp Gly Pro His Cys Val Ser Ser Cys Pro His Gly Val Leu Gly

565 570 575

Ala Lys Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln Asn Glu Cys Arg

580 585 590

Pro Cys His Glu Asn Cys Thr Gln Gly Cys Lys Gly Pro Glu Leu Gln

595 600 605

Asp Cys Leu Gly Gln Thr Leu Val Leu Ile Gly Lys Thr His Leu Thr

610 615 620

<210> 10

<211> 21

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 10

Met Ala Leu Thr Val Ile Ala Gly Leu Val Val Ile Phe Met Met Leu

1 5 10 15

Gly Gly Thr Phe Leu

20

<210> 11

<211> 678

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 11

Tyr Trp Arg Gly Arg Arg Ile Gln Asn Lys Arg Ala Met Arg Arg Tyr

1 5 10 15

Leu Glu Arg Gly Glu Ser Ile Glu Pro Leu Asp Pro Ser Glu Lys Ala

20 25 30

Asn Lys Val Leu Ala Arg Ile Phe Lys Glu Thr Glu Leu Arg Lys Leu

35 40 45

Lys Val Leu Gly Ser Gly Val Phe Gly Thr Val His Lys Gly Val Trp

50 55 60

Ile Pro Glu Gly Glu Ser Ile Lys Ile Pro Val Cys Ile Lys Val Ile

65 70 75 80

Glu Asp Lys Ser Gly Arg Gln Ser Phe Gln Ala Val Thr Asp His Met

85 90 95

Leu Ala Ile Gly Ser Leu Asp His Ala His Ile Val Arg Leu Leu Gly

100 105 110

Leu Cys Pro Gly Ser Ser Leu Gln Leu Val Thr Gln Tyr Leu Pro Leu

115 120 125

Gly Ser Leu Leu Asp His Val Arg Gln His Arg Gly Ala Leu Gly Pro

130 135 140

Gln Leu Leu Leu Asn Trp Gly Val Gln Ile Ala Lys Gly Met Tyr Tyr

145 150 155 160

Leu Glu Glu His Gly Met Val His Arg Asn Leu Ala Ala Arg Asn Val

165 170 175

Leu Leu Lys Ser Pro Ser Gln Val Gln Val Ala Asp Phe Gly Val Ala

180 185 190

Asp Leu Leu Pro Pro Asp Asp Lys Gln Leu Leu Tyr Ser Glu Ala Lys

195 200 205

Thr Pro Ile Lys Trp Met Ala Leu Glu Ser Ile His Phe Gly Lys Tyr

210 215 220

Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu

225 230 235 240

Met Thr Phe Gly Ala Glu Pro Tyr Ala Gly Leu Arg Leu Ala Glu Val

245 250 255

Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Ala Gln Pro Gln Ile Cys

260 265 270

Thr Ile Asp Val Tyr Met Val Met Val Lys Cys Trp Met Ile Asp Glu

275 280 285

Asn Ile Arg Pro Thr Phe Lys Glu Leu Ala Asn Glu Phe Thr Arg Met

290 295 300

Ala Arg Asp Pro Pro Arg Tyr Leu Val Ile Lys Arg Glu Ser Gly Pro

305 310 315 320

Gly Ile Ala Pro Gly Pro Glu Pro His Gly Leu Thr Asn Lys Lys Leu

325 330 335

Glu Glu Val Glu Leu Glu Pro Glu Leu Asp Leu Asp Leu Asp Leu Glu

340 345 350

Ala Glu Glu Asp Asn Leu Ala Thr Thr Thr Leu Gly Ser Ala Leu Ser

355 360 365

Leu Pro Val Gly Thr Leu Asn Arg Pro Arg Gly Ser Gln Ser Leu Leu

370 375 380

Ser Pro Ser Ser Gly Tyr Met Pro Met Asn Gln Gly Asn Leu Gly Glu

385 390 395 400

Ser Cys Gln Glu Ser Ala Val Ser Gly Ser Ser Glu Arg Cys Pro Arg

405 410 415

Pro Val Ser Leu His Pro Met Pro Arg Gly Cys Leu Ala Ser Glu Ser

420 425 430

Ser Glu Gly His Val Thr Gly Ser Glu Ala Glu Leu Gln Glu Lys Val

435 440 445

Ser Met Cys Arg Ser Arg Ser Arg Ser Arg Ser Pro Arg Pro Arg Gly

450 455 460

Asp Ser Ala Tyr His Ser Gln Arg His Ser Leu Leu Thr Pro Val Thr

465 470 475 480

Pro Leu Ser Pro Pro Gly Leu Glu Glu Glu Asp Val Asn Gly Tyr Val

485 490 495

Met Pro Asp Thr His Leu Lys Gly Thr Pro Ser Ser Arg Glu Gly Thr

500 505 510

Leu Ser Ser Val Gly Leu Ser Ser Val Leu Gly Thr Glu Glu Glu Asp

515 520 525

Glu Asp Glu Glu Tyr Glu Tyr Met Asn Arg Arg Arg Arg His Ser Pro

530 535 540

Pro His Pro Pro Arg Pro Ser Ser Leu Glu Glu Leu Gly Tyr Glu Tyr

545 550 555 560

Met Asp Val Gly Ser Asp Leu Ser Ala Ser Leu Gly Ser Thr Gln Ser

565 570 575

Cys Pro Leu His Pro Val Pro Ile Met Pro Thr Ala Gly Thr Thr Pro

580 585 590

Asp Glu Asp Tyr Glu Tyr Met Asn Arg Gln Arg Asp Gly Gly Gly Pro

595 600 605

Gly Gly Asp Tyr Ala Ala Met Gly Ala Cys Pro Ala Ser Glu Gln Gly

610 615 620

Tyr Glu Glu Met Arg Ala Phe Gln Gly Pro Gly His Gln Ala Pro His

625 630 635 640

Val His Tyr Ala Arg Leu Lys Thr Leu Arg Ser Leu Glu Ala Thr Asp

645 650 655

Ser Ala Phe Asp Asn Pro Asp Tyr Trp His Ser Arg Leu Phe Pro Lys

660 665 670

Ala Asn Ala Gln Arg Thr

675

<210> 12

<211> 44

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 12

Tyr Trp Arg Gly Arg Arg Ile Gln Asn Lys Arg Ala Met Arg Arg Tyr

1 5 10 15

Leu Glu Arg Gly Glu Ser Ile Glu Pro Leu Asp Pro Ser Glu Lys Ala

20 25 30

Asn Lys Val Leu Ala Arg Ile Phe Lys Glu Thr Glu

35 40

<210> 13

<211> 258

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 13

Leu Arg Lys Leu Lys Val Leu Gly Ser Gly Val Phe Gly Thr Val His

1 5 10 15

Lys Gly Val Trp Ile Pro Glu Gly Glu Ser Ile Lys Ile Pro Val Cys

20 25 30

Ile Lys Val Ile Glu Asp Lys Ser Gly Arg Gln Ser Phe Gln Ala Val

35 40 45

Thr Asp His Met Leu Ala Ile Gly Ser Leu Asp His Ala His Ile Val

50 55 60

Arg Leu Leu Gly Leu Cys Pro Gly Ser Ser Leu Gln Leu Val Thr Gln

65 70 75 80

Tyr Leu Pro Leu Gly Ser Leu Leu Asp His Val Arg Gln His Arg Gly

85 90 95

Ala Leu Gly Pro Gln Leu Leu Leu Asn Trp Gly Val Gln Ile Ala Lys

100 105 110

Gly Met Tyr Tyr Leu Glu Glu His Gly Met Val His Arg Asn Leu Ala

115 120 125

Ala Arg Asn Val Leu Leu Lys Ser Pro Ser Gln Val Gln Val Ala Asp

130 135 140

Phe Gly Val Ala Asp Leu Leu Pro Pro Asp Asp Lys Gln Leu Leu Tyr

145 150 155 160

Ser Glu Ala Lys Thr Pro Ile Lys Trp Met Ala Leu Glu Ser Ile His

165 170 175

Phe Gly Lys Tyr Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr

180 185 190

Val Trp Glu Leu Met Thr Phe Gly Ala Glu Pro Tyr Ala Gly Leu Arg

195 200 205

Leu Ala Glu Val Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Ala Gln

210 215 220

Pro Gln Ile Cys Thr Ile Asp Val Tyr Met Val Met Val Lys Cys Trp

225 230 235 240

Met Ile Asp Glu Asn Ile Arg Pro Thr Phe Lys Glu Leu Ala Asn Glu

245 250 255

Phe Thr

<210> 14

<211> 376

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 14

Arg Met Ala Arg Asp Pro Pro Arg Tyr Leu Val Ile Lys Arg Glu Ser

1 5 10 15

Gly Pro Gly Ile Ala Pro Gly Pro Glu Pro His Gly Leu Thr Asn Lys

20 25 30

Lys Leu Glu Glu Val Glu Leu Glu Pro Glu Leu Asp Leu Asp Leu Asp

35 40 45

Leu Glu Ala Glu Glu Asp Asn Leu Ala Thr Thr Thr Leu Gly Ser Ala

50 55 60

Leu Ser Leu Pro Val Gly Thr Leu Asn Arg Pro Arg Gly Ser Gln Ser

65 70 75 80

Leu Leu Ser Pro Ser Ser Gly Tyr Met Pro Met Asn Gln Gly Asn Leu

85 90 95

Gly Glu Ser Cys Gln Glu Ser Ala Val Ser Gly Ser Ser Glu Arg Cys

100 105 110

Pro Arg Pro Val Ser Leu His Pro Met Pro Arg Gly Cys Leu Ala Ser

115 120 125

Glu Ser Ser Glu Gly His Val Thr Gly Ser Glu Ala Glu Leu Gln Glu

130 135 140

Lys Val Ser Met Cys Arg Ser Arg Ser Arg Ser Arg Ser Pro Arg Pro

145 150 155 160

Arg Gly Asp Ser Ala Tyr His Ser Gln Arg His Ser Leu Leu Thr Pro

165 170 175

Val Thr Pro Leu Ser Pro Pro Gly Leu Glu Glu Glu Asp Val Asn Gly

180 185 190

Tyr Val Met Pro Asp Thr His Leu Lys Gly Thr Pro Ser Ser Arg Glu

195 200 205

Gly Thr Leu Ser Ser Val Gly Leu Ser Ser Val Leu Gly Thr Glu Glu

210 215 220

Glu Asp Glu Asp Glu Glu Tyr Glu Tyr Met Asn Arg Arg Arg Arg His

225 230 235 240

Ser Pro Pro His Pro Pro Arg Pro Ser Ser Leu Glu Glu Leu Gly Tyr

245 250 255

Glu Tyr Met Asp Val Gly Ser Asp Leu Ser Ala Ser Leu Gly Ser Thr

260 265 270

Gln Ser Cys Pro Leu His Pro Val Pro Ile Met Pro Thr Ala Gly Thr

275 280 285

Thr Pro Asp Glu Asp Tyr Glu Tyr Met Asn Arg Gln Arg Asp Gly Gly

290 295 300

Gly Pro Gly Gly Asp Tyr Ala Ala Met Gly Ala Cys Pro Ala Ser Glu

305 310 315 320

Gln Gly Tyr Glu Glu Met Arg Ala Phe Gln Gly Pro Gly His Gln Ala

325 330 335

Pro His Val His Tyr Ala Arg Leu Lys Thr Leu Arg Ser Leu Glu Ala

340 345 350

Thr Asp Ser Ala Phe Asp Asn Pro Asp Tyr Trp His Ser Arg Leu Phe

355 360 365

Pro Lys Ala Asn Ala Gln Arg Thr

370 375

<210> 15

<211> 164

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 15

Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr Leu Asn Gly

1 5 10 15

Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr Leu Tyr Lys

20 25 30

Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu Ile Val Leu

35 40 45

Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile Arg Glu Val

50 55 60

Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr Leu Pro Leu

65 70 75 80

Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp Gly Lys Phe

85 90 95

Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser His Ala Leu

100 105 110

Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser Gly Gly Val

115 120 125

Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr Ile Asp Trp

130 135 140

Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val Lys Asp Asn

145 150 155 160

Gly Arg Ser Cys

<210> 16

<211> 146

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 16

Pro Pro Cys His Glu Val Cys Lys Gly Arg Cys Trp Gly Pro Gly Ser

1 5 10 15

Glu Asp Cys Gln Thr Leu Thr Lys Thr Ile Cys Ala Pro Gln Cys Asn

20 25 30

Gly His Cys Phe Gly Pro Asn Pro Asn Gln Cys Cys His Asp Glu Cys

35 40 45

Ala Gly Gly Cys Ser Gly Pro Gln Asp Thr Asp Cys Phe Ala Cys Arg

50 55 60

His Phe Asn Asp Ser Gly Ala Cys Val Pro Arg Cys Pro Gln Pro Leu

65 70 75 80

Val Tyr Asn Lys Leu Thr Phe Gln Leu Glu Pro Asn Pro His Thr Lys

85 90 95

Tyr Gln Tyr Gly Gly Val Cys Val Ala Ser Cys Pro His Asn Phe Val

100 105 110

Val Asp Gln Thr Ser Cys Val Arg Ala Cys Pro Pro Asp Lys Met Glu

115 120 125

Val Asp Lys Asn Gly Leu Lys Met Cys Glu Pro Cys Gly Gly Leu Cys

130 135 140

Pro Lys

145

<210> 17

<211> 166

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 17

Ala Cys Glu Gly Thr Gly Ser Gly Ser Arg Phe Gln Thr Val Asp Ser

1 5 10 15

Ser Asn Ile Asp Gly Phe Val Asn Cys Thr Lys Ile Leu Gly Asn Leu

20 25 30

Asp Phe Leu Ile Thr Gly Leu Asn Gly Asp Pro Trp His Lys Ile Pro

35 40 45

Ala Leu Asp Pro Glu Lys Leu Asn Val Phe Arg Thr Val Arg Glu Ile

50 55 60

Thr Gly Tyr Leu Asn Ile Gln Ser Trp Pro Pro His Met His Asn Phe

65 70 75 80

Ser Val Phe Ser Asn Leu Thr Thr Ile Gly Gly Arg Ser Leu Tyr Asn

85 90 95

Arg Gly Phe Ser Leu Leu Ile Met Lys Asn Leu Asn Val Thr Ser Leu

100 105 110

Gly Phe Arg Ser Leu Lys Glu Ile Ser Ala Gly Arg Ile Tyr Ile Ser

115 120 125

Ala Asn Arg Gln Leu Cys Tyr His His Ser Leu Asn Trp Thr Lys Val

130 135 140

Leu Arg Gly Pro Thr Glu Glu Arg Leu Asp Ile Lys His Asn Arg Pro

145 150 155 160

Arg Arg Asp Cys Val Ala

165

<210> 18

<211> 148

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 18

Glu Gly Lys Val Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys Trp Gly

1 5 10 15

Pro Gly Pro Gly Gln Cys Leu Ser Cys Arg Asn Tyr Ser Arg Gly Gly

20 25 30

Val Cys Val Thr His Cys Asn Phe Leu Asn Gly Glu Pro Arg Glu Phe

35 40 45

Ala His Glu Ala Glu Cys Phe Ser Cys His Pro Glu Cys Gln Pro Met

50 55 60

Glu Gly Thr Ala Thr Cys Asn Gly Ser Gly Ser Asp Thr Cys Ala Gln

65 70 75 80

Cys Ala His Phe Arg Asp Gly Pro His Cys Val Ser Ser Cys Pro His

85 90 95

Gly Val Leu Gly Ala Lys Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln

100 105 110

Asn Glu Cys Arg Pro Cys His Glu Asn Cys Thr Gln Gly Cys Lys Gly

115 120 125

Pro Glu Leu Gln Asp Cys Leu Gly Gln Thr Leu Val Leu Ile Gly Lys

130 135 140

Thr His Leu Thr

145

<210> 19

<211> 17

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 19

Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu Glu Pro Asn Pro

1 5 10 15

His

<210> 20

<211> 1283

<212> PRT

<213> Kiwi berry (Rhesus macaque)

<400> 20

Met Gly Asn Leu Glu Ile Val Leu Thr Gly His Asn Ala Asp Leu Ser

1 5 10 15

Phe Leu Gln Trp Ile Arg Glu Val Thr Gly Tyr Val Leu Val Ala Met

20 25 30

Asn Glu Phe Ser Thr Leu Pro Leu Pro Asn Leu Arg Val Val Arg Gly

35 40 45

Thr Gln Val Tyr Asp Gly Lys Phe Ala Ile Phe Val Met Leu Asn Tyr

50 55 60

Asn Thr Asn Ser Ser His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu

65 70 75 80

Thr Glu Ile Leu Ser Gly Gly Val Tyr Ile Glu Lys Asn Asp Lys Leu

85 90 95

Cys His Met Asp Thr Ile Asp Trp Lys Asp Ile Val Arg Asp Gln Asp

100 105 110

Ala Glu Ile Val Val Lys Asp Asn Gly Arg Ser Cys Pro Leu Cys His

115 120 125

Glu Val Cys Lys Gly Arg Cys Trp Gly Pro Gly Pro Glu Asp Cys Gln

130 135 140

Thr Leu Thr Lys Thr Ile Cys Ala Pro Gln Cys Asn Gly His Cys Phe

145 150 155 160

Gly Pro Asn Pro Asn Gln Cys Cys His Asp Glu Cys Ala Gly Gly Cys

165 170 175

Ser Gly Pro Gln Asp Thr Asp Cys Phe Ala Cys Arg His Phe Asn Asp

180 185 190

Ser Gly Ala Cys Val Pro Arg Cys Pro Gln Pro Leu Val Tyr Asn Lys

195 200 205

Leu Thr Phe Gln Leu Glu Pro Asn Pro His Thr Lys Tyr Gln Tyr Gly

210 215 220

Gly Val Cys Val Ala Ser Cys Pro His Asn Phe Val Val Asp Gln Thr

225 230 235 240

Ser Cys Val Arg Ala Cys Pro Pro Asp Lys Met Glu Val Asp Lys Asn

245 250 255

Gly Leu Lys Met Cys Glu Pro Cys Gly Gly Leu Cys Pro Lys Ala Cys

260 265 270

Glu Gly Thr Gly Ser Gly Ser Arg Phe Gln Thr Val Asp Ser Ser Asn

275 280 285

Ile Asp Gly Phe Val Asn Cys Thr Lys Ile Leu Gly Asn Leu Asp Phe

290 295 300

Leu Ile Thr Gly Leu Asn Gly Asp Pro Trp His Lys Ile Pro Ala Leu

305 310 315 320

Asp Pro Glu Lys Leu Asn Val Phe Arg Thr Val Arg Glu Ile Thr Gly

325 330 335

Tyr Leu Asn Ile Gln Ser Trp Pro Pro His Met Tyr Asn Phe Ser Val

340 345 350

Phe Ser Asn Leu Thr Thr Ile Gly Gly Arg Ser Leu Tyr Asn Arg Gly

355 360 365

Phe Ser Leu Leu Ile Met Lys Asn Leu Asn Val Thr Ser Leu Gly Phe

370 375 380

Arg Ser Leu Lys Glu Ile Ser Ala Gly Arg Ile Tyr Ile Ser Ala Asn

385 390 395 400

Arg Gln Leu Cys Tyr His His Ser Leu Asn Trp Thr Lys Val Leu Arg

405 410 415

Gly Pro Thr Glu Glu Arg Leu Asp Ile Lys His Asn Arg Pro Arg Arg

420 425 430

Asp Cys Val Ala Glu Gly Lys Val Cys Asp Pro Leu Cys Ser Ser Gly

435 440 445

Gly Cys Trp Gly Pro Gly Pro Gly Gln Cys Leu Ser Cys Arg Asn Tyr

450 455 460

Ser Arg Gly Gly Val Cys Val Thr His Cys Asn Phe Leu Asn Gly Glu

465 470 475 480

Pro Arg Glu Phe Ala His Glu Ala Glu Cys Phe Ser Cys His Pro Glu

485 490 495

Cys Gln Pro Met Glu Gly Thr Ala Thr Cys Asn Gly Ser Gly Ser Asp

500 505 510

Thr Cys Ala Gln Cys Ala His Phe Arg Asp Gly Pro His Cys Val Ser

515 520 525

Ser Cys Pro His Gly Val Leu Gly Ala Lys Gly Pro Ile Tyr Lys Tyr

530 535 540

Pro Asp Val Gln Asn Glu Cys Arg Pro Cys His Glu Asn Cys Thr Gln

545 550 555 560

Gly Cys Lys Gly Pro Glu Leu Gln Asp Cys Leu Gly Gln Thr Leu Val

565 570 575

Leu Ile Gly Lys Thr His Leu Thr Met Ala Leu Thr Val Ile Ala Gly

580 585 590

Leu Val Val Ile Phe Met Met Leu Gly Gly Thr Phe Leu Tyr Trp Arg

595 600 605

Gly Arg Arg Ile Gln Asn Lys Arg Ala Met Arg Arg Tyr Leu Glu Arg

610 615 620

Gly Glu Ser Ile Glu Pro Leu Asp Pro Ser Glu Lys Ala Asn Lys Val

625 630 635 640

Leu Ala Arg Ile Phe Lys Glu Thr Glu Leu Arg Lys Leu Lys Val Leu

645 650 655

Gly Ser Gly Val Phe Gly Thr Val His Lys Gly Val Trp Ile Pro Glu

660 665 670

Gly Glu Ser Ile Lys Ile Pro Val Cys Ile Lys Ile Ile Glu Asp Lys

675 680 685

Ser Gly Arg Gln Ser Phe Gln Ala Val Thr Asp His Met Leu Ala Ile

690 695 700

Gly Ser Leu Asp His Ala His Ile Val Arg Leu Leu Gly Leu Cys Pro

705 710 715 720

Gly Ser Ser Leu Gln Leu Val Thr Gln Tyr Leu Pro Leu Gly Ser Leu

725 730 735

Leu Asp His Val Arg Gln His Arg Gly Ala Leu Gly Pro Gln Leu Leu

740 745 750

Leu Asn Trp Gly Val Gln Ile Ala Lys Gly Met Tyr Tyr Leu Glu Glu

755 760 765

His Gly Met Val His Arg Asn Leu Ala Ala Arg Asn Val Leu Leu Lys

770 775 780

Ser Pro Ser Gln Val Gln Val Ala Asp Phe Gly Val Ala Asp Leu Leu

785 790 795 800

Pro Pro Asp Asp Lys Gln Leu Leu Tyr Ser Glu Ala Lys Thr Pro Ile

805 810 815

Lys Trp Met Ala Leu Glu Ser Ile His Phe Gly Lys Tyr Thr His Gln

820 825 830

Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe

835 840 845

Gly Ala Glu Pro Tyr Ala Gly Leu Arg Leu Ala Glu Val Pro Asp Leu

850 855 860

Leu Glu Lys Gly Glu Arg Leu Ala Gln Pro Gln Ile Cys Thr Ile Asp

865 870 875 880

Val Tyr Met Val Met Val Lys Cys Trp Met Ile Asp Glu Asn Ile Arg

885 890 895

Pro Thr Phe Lys Glu Leu Ala Asn Glu Phe Thr Arg Met Ala Arg Asp

900 905 910

Pro Pro Arg Tyr Leu Val Ile Lys Arg Glu Ser Gly Pro Gly Ile Ala

915 920 925

Pro Gly Pro Glu Pro His Gly Leu Thr Asn Lys Lys Leu Glu Glu Val

930 935 940

Glu Leu Glu Pro Glu Leu Asp Leu Asp Leu Asp Leu Glu Ala Glu Glu

945 950 955 960

Asp Asn Leu Ala Thr Thr Thr Leu Gly Ser Ala Leu Ser Leu Pro Val

965 970 975

Gly Thr Leu Asn Arg Pro Arg Gly Ser Gln Ser Leu Leu Ser Pro Ser

980 985 990

Ser Gly Tyr Met Pro Met Asn Gln Gly Asn Leu Gly Glu Ala Phe Gln

995 1000 1005

Glu Ser Ala Val Ser Gly Ser Ser Glu Trp Cys Pro Arg Pro Val

1010 1015 1020

Ser Leu His Pro Met Pro Arg Gly Cys Leu Ala Ser Glu Ser Ser

1025 1030 1035

Glu Gly His Val Thr Gly Ser Glu Ala Glu Leu Gln Glu Lys Val

1040 1045 1050

Ser Thr Cys Arg Ser Arg Ser Arg Ser Arg Ser Pro Arg Pro Arg

1055 1060 1065

Gly Asp Ser Ala Tyr His Ser Gln Arg His Ser Leu Leu Thr Pro

1070 1075 1080

Val Thr Pro Leu Ser Pro Pro Gly Leu Glu Glu Glu Asp Val Asn

1085 1090 1095

Gly Tyr Val Met Pro Asp Thr His Leu Lys Gly Thr Pro Ser Ser

1100 1105 1110

Arg Glu Gly Thr Leu Ser Ser Val Gly Leu Ser Ser Val Leu Gly

1115 1120 1125

Thr Glu Glu Glu Asp Glu Asp Glu Glu Tyr Glu Tyr Met Asn Arg

1130 1135 1140

Arg Arg Arg His Ser Pro Pro Arg Pro Pro Arg Pro Ser Ser Leu

1145 1150 1155

Glu Glu Leu Gly Tyr Glu Tyr Met Asp Val Gly Ser Asp Leu Ser

1160 1165 1170

Ala Ser Leu Gly Ser Thr Gln Ser Cys Pro Leu His Pro Val Pro

1175 1180 1185

Val Met Pro Thr Ala Gly Thr Thr Pro Asp Glu Asp Tyr Glu Tyr

1190 1195 1200

Met Asn Arg Gln Arg Gly Gly Ser Gly Pro Gly Gly Asp Tyr Ala

1205 1210 1215

Ala Met Gly Ala Cys Pro Ala Ser Glu Gln Gly Tyr Glu Glu Met

1220 1225 1230

Arg Ala Phe Gln Gly Pro Gly His Gln Ala Pro His Val His Tyr

1235 1240 1245

Ala His Leu Lys Thr Leu Arg Ser Leu Glu Ala Thr Asp Ser Ala

1250 1255 1260

Phe Asp Asn Pro Asp Tyr Trp His Ser Arg Leu Phe Pro Lys Ala

1265 1270 1275

Asn Ala Gln Arg Thr

1280

<210> 21

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Tyr Asn Lys Leu Thr Phe Gln Leu Glu Pro Asn Pro His

1 5 10

<210> 22

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Pro Arg Cys Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe

1 5 10

<210> 23

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Pro Arg Cys Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu

1 5 10 15

Glu Pro Asn Pro His

20

<210> 24

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Asp Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 25

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Thr Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 26

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 27

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 28

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 29

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 30

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 31

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile Arg Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Gly Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 32

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile Arg Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Gly Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 33

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 33

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile Arg Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Gly Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Glu Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 34

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 34

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile Arg Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Gly Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 35

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 36

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile Arg Tyr Ser Gly Gly Thr Asp Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Leu Val Thr Ile Ser Ala Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Trp Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 37

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 37

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Phe Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile Arg Tyr Ser Gly Gly Thr Asp Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Leu Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Trp Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 38

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 38

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Tyr Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile Arg Tyr Ser Gly Gly Thr Asp Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Leu Ala Thr Ile Ser Ala Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Trp Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Val Thr Val Ser Ser

115 120

<210> 39

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 39

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Thr Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 40

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 40

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 41

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 41

Gly Tyr Ser Ile Thr Ser Gly Tyr Ser

1 5

<210> 42

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 42

Gly Tyr Tyr Ile Thr Ser Gly Tyr Ser

1 5

<210> 43

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 43

Gly Tyr Xaa Ile Thr Ser Gly Tyr Ser

1 5

<210> 44

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 44

Ile His Tyr Ser Gly Gly Thr

1 5

<210> 45

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 45

Ile Arg Tyr Ser Gly Gly Thr

1 5

<210> 46

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 46

Ile Xaa Tyr Ser Gly Gly Thr

1 5

<210> 47

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 47

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr

1 5 10

<210> 48

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 48

Ala Arg Met Thr Thr Ala Pro Trp Tyr Pro Phe Asp Tyr

1 5 10

<210> 49

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 49

Ala Arg Glu Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr

1 5 10

<210> 50

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 50

Ala Arg Gly Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr

1 5 10

<210> 51

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 51

Ala Arg Xaa Thr Thr Ala Pro Xaa Tyr Pro Phe Asp Tyr

1 5 10

<210> 52

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 52

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr

20 25

<210> 53

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 53

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser

20 25

<210> 54

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 54

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Phe Leu Thr Cys Thr Val Ser

20 25

<210> 55

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 55

Asp Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr

20 25

<210> 56

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 56

Trp His Trp Ile Arg Gln Phe Pro Gly Asn Gly Leu Glu Trp Ile Gly

1 5 10 15

Ser

<210> 57

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 57

Trp His Trp Ile Arg Gln Phe Pro Gly Lys Gly Leu Glu Trp Ile Gly

1 5 10 15

Ser

<210> 58

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 58

Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Gly

1 5 10 15

Ser

<210> 59

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 59

Trp His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly

1 5 10 15

Ser

<210> 60

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 60

Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

1 5 10 15

Ser

<210> 61

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 61

Asn Tyr Asn Pro Thr Leu Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr

1 5 10 15

Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp

20 25 30

Thr Ala Val Tyr Phe Cys

35

<210> 62

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 62

Asn Tyr Asn Pro Ser Leu Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr

1 5 10 15

Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp

20 25 30

Thr Ala Val Tyr Phe Cys

35

<210> 63

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 63

Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Arg Asp Thr

1 5 10 15

Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 64

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 64

Asn Tyr Asn Pro Ser Leu Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr

1 5 10 15

Ser Lys Asn Gln Phe Ser Leu Lys Leu Gly Ser Val Thr Ala Ala Asp

20 25 30

Thr Ala Val Tyr Phe Cys

35

<210> 65

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 65

Asn Tyr Asn Pro Ser Leu Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr

1 5 10 15

Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp

20 25 30

Thr Ala Val Tyr Phe Cys

35

<210> 66

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 66

Asp Tyr Asn Pro Ser Leu Lys Ser Leu Val Thr Ile Ser Ala Asp Thr

1 5 10 15

Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 67

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 67

Asp Tyr Asn Pro Ser Leu Lys Ser Leu Val Thr Ile Ser Val Asp Thr

1 5 10 15

Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 68

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 68

Asp Tyr Asn Pro Ser Leu Lys Ser Leu Ala Thr Ile Ser Ala Asp Thr

1 5 10 15

Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 69

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 69

Asn Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr

1 5 10 15

Ser Lys Asn Gln Phe Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp

20 25 30

Thr Ala Thr Tyr Phe Cys

35

<210> 70

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 70

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 71

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 71

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

1 5 10

<210> 72

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 72

Trp Gly Gln Gly Thr Ala Val Thr Val Ser Ser

1 5 10

<210> 73

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 73

Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

1 5 10

<210> 74

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 74

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Ala

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 75

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 75

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Leu Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Met Lys Pro Gly Lys Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Phe Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Glu Tyr Phe Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys

100 105

<210> 76

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 76

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Gly Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Tyr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Ser Asp Val Pro Ser Arg Phe Ser Ala

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Glu Tyr Phe Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys

100 105

<210> 77

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 77

Val Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Pro Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Gly Tyr Ser Asp Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Glu Tyr Phe Cys Gln Gln Tyr Ser Thr His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys

100 105

<210> 78

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 78

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Gly Tyr Ser Asp Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Arg Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys

100 105

<210> 79

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 79

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Arg Tyr Gln Tyr Ser Gly Val Pro Phe Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys

100 105

<210> 80

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 80

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Arg Tyr Gln Tyr Ser Gly Val Pro Phe Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys

100 105

<210> 81

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 81

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Arg Tyr Gln Tyr Ser Gly Val Pro Phe Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys

100 105

<210> 82

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 82

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Met Pro Gly Lys Ser Pro Glu Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Ser Asp Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Met Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys

100 105

<210> 83

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 83

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 84

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 84

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Phe Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Ser Ser Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Met Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Thr Thr His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys

100 105

<210> 85

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 85

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Met Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Gly Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 86

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 86

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Leu Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Met Lys Leu Gly Lys Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Phe Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Glu Tyr Phe Cys Gln Gln Tyr Phe Ser His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys

100 105

<210> 87

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 87

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Ser Asp Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Met Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys

100 105

<210> 88

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 88

Gln Ile Val Gly Ser Asn

1 5

<210> 89

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 89

Gln Ile Val Gly Tyr Asn

1 5

<210> 90

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 90

Gln Ile Val Gly Pro Asn

1 5

<210> 91

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 91

Gln Ile Val Gly Xaa Asn

1 5

<210> 92

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 92

Ser Ala Ser

1

<210> 93

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 93

Ser Ala Arg

1

<210> 94

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 94

Ser Ala Xaa

1

<210> 95

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 95

Gln Gln Tyr Ser Ser His Pro Leu Thr

1 5

<210> 96

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 96

Gln Gln Tyr Ser Thr His Pro Leu Thr

1 5

<210> 97

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 97

Gln Gln Tyr Thr Thr His Pro Leu Thr

1 5

<210> 98

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 98

Gln Gln Tyr Phe Ser His Pro Leu Thr

1 5

<210> 99

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 99

Gln Gln Tyr Xaa Xaa His Pro Leu Thr

1 5

<210> 100

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 100

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Leu Val Thr Ile Thr Cys Lys Ala Ser

20 25

<210> 101

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 101

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Gly Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

20 25

<210> 102

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 102

Val Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

20 25

<210> 103

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 103

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

20 25

<210> 104

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 104

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

20 25

<210> 105

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 105

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Phe Thr Cys Lys Ala Ser

20 25

<210> 106

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 106

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser

20 25

<210> 107

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 107

Val Ala Trp Tyr Gln Met Lys Pro Gly Lys Ser Pro Lys Pro Leu Ile

1 5 10 15

Tyr

<210> 108

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 108

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Pro Leu Ile

1 5 10 15

Tyr

<210> 109

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 109

Val Ala Trp Tyr Gln Gln Met Pro Gly Lys Ser Pro Glu Pro Leu Ile

1 5 10 15

Tyr

<210> 110

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 110

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Pro Leu Ile

1 5 10 15

Tyr

<210> 111

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 111

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Met Pro Leu Ile

1 5 10 15

Tyr

<210> 112

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 112

Val Ala Trp Tyr Gln Met Lys Leu Gly Lys Ser Pro Lys Pro Leu Ile

1 5 10 15

Tyr

<210> 113

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 113

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile

1 5 10 15

Tyr

<210> 114

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 114

Tyr Leu Tyr Phe Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala

20 25 30

Glu Tyr Phe Cys

35

<210> 115

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 115

Tyr Leu Tyr Ser Asp Val Pro Ser Arg Phe Ser Ala Ser Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala

20 25 30

Glu Tyr Phe Cys

35

<210> 116

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 116

Tyr Gly Tyr Ser Asp Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala

20 25 30

Glu Tyr Phe Cys

35

<210> 117

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 117

Tyr Gly Tyr Ser Asp Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Arg Pro Glu Asp Val Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 118

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 118

Tyr Gln Tyr Ser Gly Val Pro Phe Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 119

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 119

Tyr Leu Tyr Ser Asp Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Met Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 120

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 120

Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 121

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 121

Tyr Leu Tyr Ser Ser Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Thr Glu Phe Thr Met Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 122

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 122

Tyr Gly Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 123

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 123

Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Ala Ser Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser Glu Asp Leu Ala

20 25 30

Glu Tyr Phe Cys

35

<210> 124

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 124

Phe Gly Pro Gly Thr Lys Val Glu Ile Lys

1 5 10

<210> 125

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 125

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210> 126

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 126

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

1 5 10

<210> 127

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 127

Glu Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr

20 25 30

Asn Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly His Ile Asn Pro Tyr Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe

65 70 75 80

Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Val Ser Leu Arg Trp Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 128

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 128

Gly Tyr Ser Phe Thr Asp Tyr Asn

1 5

<210> 129

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 129

Ile Asn Pro Tyr Asn Gly Gly Thr

1 5

<210> 130

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 130

Val Ser Leu Arg Trp Gly Ala Met Asp Tyr

1 5 10

<210> 131

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 131

Glu Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser

20 25

<210> 132

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 132

Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly

1 5 10 15

His

<210> 133

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 133

Thr Tyr Asn Gln Lys Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Lys

1 5 10 15

Ser Ser Ser Thr Ala Phe Met His Leu Asn Ser Leu Thr Ser Glu Asp

20 25 30

Ser Ala Val Tyr Phe Cys

35

<210> 134

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 134

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

1 5 10

<210> 135

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 135

Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Ser Asn Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 136

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 136

Ser Ser Val Ser Tyr

1 5

<210> 137

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 137

Leu Thr Ser

1

<210> 138

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 138

Gln Gln Trp Asn Ser Asn Pro Tyr Thr

1 5

<210> 139

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 139

Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser

20 25

<210> 140

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 140

Met Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile

1 5 10 15

Tyr

<210> 141

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 141

Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 142

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 142

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210> 143

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 143

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Lys Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Ile Asp Pro Ala Asn Gly Asn Thr Asn Tyr Asp Pro Lys

50 55 60

Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala

65 70 75 80

Tyr Leu Gln Leu Ser Ser Leu Ser Ser Glu Asp Thr Ala Val Tyr Phe

85 90 95

Cys Ala Arg Gly Leu His Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

100 105 110

Ser

<210> 144

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 144

Gly Phe Asn Ile Lys Asp Thr Tyr

1 5

<210> 145

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 145

Ile Asp Pro Ala Asn Gly Asn Thr

1 5

<210> 146

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 146

Ala Arg Gly Leu His

1 5

<210> 147

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 147

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser

20 25

<210> 148

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 148

Ile His Trp Val Lys Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile Gly

1 5 10 15

Lys

<210> 149

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 149

Asn Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr

1 5 10 15

Ser Ser Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Ser Ser Glu Asp

20 25 30

Thr Ala Val Tyr Phe Cys

35

<210> 150

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 150

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 151

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 151

Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr

1 5 10

<210> 152

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 152

Leu Ala Ser

1

<210> 153

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 153

Gln His Ser Arg Glu Leu Pro Tyr Thr

1 5

<210> 154

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 154

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser

20 25

<210> 155

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 155

Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

1 5 10 15

Tyr

<210> 156

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 156

Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 157

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 157

Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Asn Phe Ile Thr Ser Gly

20 25 30

Tyr Phe Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Phe Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Lys Pro Ser Leu

50 55 60

Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Glu Asn Tyr Gly Phe Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 158

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 158

Gly Asn Phe Ile Thr Ser Gly Tyr Phe

1 5

<210> 159

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 159

Ile Ser Tyr Asp Gly Ser Asn

1 5

<210> 160

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 160

Ala Arg Glu Asn Tyr Gly Phe Gly Phe Asp Tyr

1 5 10

<210> 161

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 161

Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr

20 25

<210> 162

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 162

Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Gly

1 5 10 15

Phe

<210> 163

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 163

Asn Tyr Lys Pro Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr

1 5 10 15

Ser Lys Asn Gln Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp

20 25 30

Thr Ala Thr Tyr Tyr Cys

35

<210> 164

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 164

Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Pro Val Ser Ile Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Asp Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Trp Lys Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Phe Thr Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 165

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 165

Gln Ser Leu Leu Tyr Ser Asp Asn Gln Lys Asn Tyr

1 5 10

<210> 166

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 166

Trp Ala Ser

1

<210> 167

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 167

Gln Gln Tyr Phe Thr Phe Pro Trp Thr

1 5

<210> 168

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 168

Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Pro Val Ser Ile Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser

20 25

<210> 169

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 169

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

1 5 10 15

Tyr

<210> 170

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 170

Thr Trp Lys Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala

20 25 30

Val Tyr Tyr Cys

35

<210> 171

<211> 330

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 171

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 172

<211> 98

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 172

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val

<210> 173

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 173

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

1 5 10

<210> 174

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 174

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

1 5 10 15

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

20 25 30

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

35 40 45

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

50 55 60

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

65 70 75 80

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

85 90 95

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

100 105 110

Lys

<210> 175

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 175

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

1 5 10 15

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

65 70 75 80

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

100 105

<210> 176

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 176

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

1 5 10 15

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

65 70 75 80

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

100 105

<210> 177

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 177

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 178

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 178

Met Lys Val Leu Ser Leu Leu Tyr Leu Leu Thr Ala Ile Pro Gly Ile

1 5 10 15

Leu Ser

<210> 179

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 179

Met Arg Val Leu Ile Leu Leu Cys Leu Phe Thr Ala Phe Pro Gly Ile

1 5 10 15

Leu Ser

<210> 180

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 180

Met Glu Ser Gln Thr Gln Val Phe Val Tyr Met Leu Leu Trp Leu Ser

1 5 10 15

Gly Val Asp Gly

20

<210> 181

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 181

Met Glu Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Thr Gly

1 5 10 15

Val His Ser

<210> 182

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 182

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Met Ser Ala Ser

1 5 10 15

Val Met Met Ser Arg Gly

20

<210> 183

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 183

Met Lys Cys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly

1 5 10 15

Val Asn Ser

<210> 184

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 184

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly

20

<210> 185

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 185

Met Glu Leu Gly Leu Arg Trp Val Phe Leu Ile Ala Thr Leu Ala Gly

1 5 10 15

Ala Arg Cys

<210> 186

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 186

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys

20

<210> 187

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 187

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Thr Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 188

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 188

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Leu Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Met Lys Pro Gly Lys Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Phe Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Glu Tyr Phe Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 189

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 189

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 190

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 190

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Gly Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Tyr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Ser Asp Val Pro Ser Arg Phe Ser Ala

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Glu Tyr Phe Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 191

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 191

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 192

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 192

Val Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Pro Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Gly Tyr Ser Asp Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Glu Tyr Phe Cys Gln Gln Tyr Ser Thr His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 193

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 193

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 194

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 194

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 195

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 195

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Gly Tyr Ser Asp Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Arg Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 196

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 196

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 197

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 197

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile Arg Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Gly Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 198

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 198

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile Arg Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Gly Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 199

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 199

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Arg Tyr Gln Tyr Ser Gly Val Pro Phe Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 200

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 200

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile Arg Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Gly Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Glu Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 201

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 201

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Arg Tyr Gln Tyr Ser Gly Val Pro Phe Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 202

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 202

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile Arg Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Gly Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 203

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 203

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Arg Tyr Gln Tyr Ser Gly Val Pro Phe Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 204

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 204

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 205

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 205

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Met Pro Gly Lys Ser Pro Glu Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Ser Asp Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Met Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 206

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 206

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile Arg Tyr Ser Gly Gly Thr Asp Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Leu Val Thr Ile Ser Ala Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Trp Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 207

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 207

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 208

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 208

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Phe Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile Arg Tyr Ser Gly Gly Thr Asp Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Leu Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Trp Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 209

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 209

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Phe Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Ser Ser Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Met Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Thr Thr His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 210

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 210

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Tyr Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile Arg Tyr Ser Gly Gly Thr Asp Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Leu Ala Thr Ile Ser Ala Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Trp Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ala Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 211

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 211

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Met Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Gly Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 212

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 212

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Thr Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 213

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 213

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Leu Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Met Lys Leu Gly Lys Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Phe Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Glu Tyr Phe Cys Gln Gln Tyr Phe Ser His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 214

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 214

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 215

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 215

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Ser Asp Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Met Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 216

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 216

Asp Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 217

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 217

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Ile Val Gly Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Ala

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Ser Ser His Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 218

<211> 447

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 218

Glu Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr

20 25 30

Asn Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly His Ile Asn Pro Tyr Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe

65 70 75 80

Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Val Ser Leu Arg Trp Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 219

<211> 213

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 219

Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Ser Asn Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 220

<211> 443

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 220

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Lys Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Ile Asp Pro Ala Asn Gly Asn Thr Asn Tyr Asp Pro Lys

50 55 60

Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala

65 70 75 80

Tyr Leu Gln Leu Ser Ser Leu Ser Ser Glu Asp Thr Ala Val Tyr Phe

85 90 95

Cys Ala Arg Gly Leu His Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

100 105 110

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

115 120 125

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

180 185 190

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

210 215 220

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

340 345 350

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210> 221

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 221

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 222

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 222

Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Asn Phe Ile Thr Ser Gly

20 25 30

Tyr Phe Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Phe Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Lys Pro Ser Leu

50 55 60

Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Glu Asn Tyr Gly Phe Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 223

<211> 220

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 223

Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Pro Val Ser Ile Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Asp Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Trp Lys Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Phe Thr Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 224

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 224

Asp Val Gln Leu Gln Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr

20 25

<210> 225

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 225

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile Arg Tyr Ser Gly Gly Thr Asp Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Leu Val Thr Ile Ser Ala Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Trp Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly

225 230 235 240

Pro Asp Val Phe Cys Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu

325 330 335

Cys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 226

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 226

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile Arg Tyr Ser Gly Gly Thr Asp Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Leu Val Thr Ile Ser Ala Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Trp Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gln Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 227

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 227

Asp Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly

225 230 235 240

Pro Asp Val Phe Cys Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu

325 330 335

Cys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 228

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 228

Asp Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Ser Ile His Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Met Thr Thr Ala Pro Arg Tyr Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly

225 230 235 240

Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 229

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 229

Cys Phe Gly Pro Asn Pro Asn Gln Cys Cys His Asp Glu Cys Ala Gly

1 5 10 15

Gly Cys

<210> 230

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 230

Pro Asn Pro Asn Gln

1 5

<210> 231

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 231

Asp Glu Cys Ala Gly

1 5

<210> 232

<211> 640

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 232

Met Ser Glu Arg Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys

1 5 10 15

Glu Arg Gly Ser Gly Lys Lys Pro Glu Ser Ala Ala Gly Ser Gln Ser

20 25 30

Pro Ala Leu Pro Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala

35 40 45

Ala Gly Ser Lys Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser

50 55 60

Ser Leu Arg Phe Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys

65 70 75 80

Asn Lys Pro Gln Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu

85 90 95

Leu Arg Ile Asn Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys

100 105 110

Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr

115 120 125

Ile Val Glu Ser Asn Glu Ile Ile Thr Gly Met Pro Ala Ser Thr Glu

130 135 140

Gly Ala Tyr Val Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr

145 150 155 160

Glu Gly Ala Asn Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr

165 170 175

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

180 185 190

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

195 200 205

Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn

210 215 220

Val Pro Met Lys Val Gln Asn Gln Glu Lys Ala Glu Glu Leu Tyr Gln

225 230 235 240

Lys Arg Val Leu Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val Val

245 250 255

Gly Ile Met Cys Val Val Ala Tyr Cys Lys Thr Lys Lys Gln Arg Lys

260 265 270

Lys Leu His Asp Arg Leu Arg Gln Ser Leu Arg Ser Glu Arg Asn Asn

275 280 285

Met Met Asn Ile Ala Asn Gly Pro His His Pro Asn Pro Pro Pro Glu

290 295 300

Asn Val Gln Leu Val Asn Gln Tyr Val Ser Lys Asn Val Ile Ser Ser

305 310 315 320

Glu His Ile Val Glu Arg Glu Ala Glu Thr Ser Phe Ser Thr Ser His

325 330 335

Tyr Thr Ser Thr Ala His His Ser Thr Thr Val Thr Gln Thr Pro Ser

340 345 350

His Ser Trp Ser Asn Gly His Thr Glu Ser Ile Leu Ser Glu Ser His

355 360 365

Ser Val Ile Val Met Ser Ser Val Glu Asn Ser Arg His Ser Ser Pro

370 375 380

Thr Gly Gly Pro Arg Gly Arg Leu Asn Gly Thr Gly Gly Pro Arg Glu

385 390 395 400

Cys Asn Ser Phe Leu Arg His Ala Arg Glu Thr Pro Asp Ser Tyr Arg

405 410 415

Asp Ser Pro His Ser Glu Arg Tyr Val Ser Ala Met Thr Thr Pro Ala

420 425 430

Arg Met Ser Pro Val Asp Phe His Thr Pro Ser Ser Pro Lys Ser Pro

435 440 445

Pro Ser Glu Met Ser Pro Pro Val Ser Ser Met Thr Val Ser Met Pro

450 455 460

Ser Met Ala Val Ser Pro Phe Met Glu Glu Glu Arg Pro Leu Leu Leu

465 470 475 480

Val Thr Pro Pro Arg Leu Arg Glu Lys Lys Phe Asp His His Pro Gln

485 490 495

Gln Phe Ser Ser Phe His His Asn Pro Ala His Asp Ser Asn Ser Leu

500 505 510

Pro Ala Ser Pro Leu Arg Ile Val Glu Asp Glu Glu Tyr Glu Thr Thr

515 520 525

Gln Glu Tyr Glu Pro Ala Gln Glu Pro Val Lys Lys Leu Ala Asn Ser

530 535 540

Arg Arg Ala Lys Arg Thr Lys Pro Asn Gly His Ile Ala Asn Arg Leu

545 550 555 560

Glu Val Asp Ser Asn Thr Ser Ser Gln Ser Ser Asn Ser Glu Ser Glu

565 570 575

Thr Glu Asp Glu Arg Val Gly Glu Asp Thr Pro Phe Leu Gly Ile Gln

580 585 590

Asn Pro Leu Ala Ala Ser Leu Glu Ala Thr Pro Ala Phe Arg Leu Ala

595 600 605

Asp Ser Arg Thr Asn Pro Ala Gly Arg Phe Ser Thr Gln Glu Glu Ile

610 615 620

Gln Ala Arg Leu Ser Ser Val Ile Ala Asn Gln Asp Pro Ile Ala Val

625 630 635 640

<210> 233

<211> 45

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 233

His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly

1 5 10 15

Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu

20 25 30

Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr

35 40 45

<210> 234

<211> 850

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 234

Met Arg Gln Val Cys Cys Ser Ala Leu Pro Pro Pro Pro Leu Glu Lys

1 5 10 15

Gly Arg Cys Ser Ser Tyr Ser Asp Ser Ser Ser Ser Ser Ser Glu Arg

20 25 30

Ser Ser Ser Ser Ser Ser Ser Ser Ser Glu Ser Gly Ser Ser Ser Arg

35 40 45

Ser Ser Ser Asn Asn Ser Ser Ile Ser Arg Pro Ala Ala Pro Pro Glu

50 55 60

Pro Arg Pro Gln Gln Gln Pro Gln Pro Arg Ser Pro Ala Ala Arg Arg

65 70 75 80

Ala Ala Ala Arg Ser Arg Ala Ala Ala Ala Gly Gly Met Arg Arg Asp

85 90 95

Pro Ala Pro Gly Phe Ser Met Leu Leu Phe Gly Val Ser Leu Ala Cys

100 105 110

Tyr Ser Pro Ser Leu Lys Ser Val Gln Asp Gln Ala Tyr Lys Ala Pro

115 120 125

Val Val Val Glu Gly Lys Val Gln Gly Leu Val Pro Ala Gly Gly Ser

130 135 140

Ser Ser Asn Ser Thr Arg Glu Pro Pro Ala Ser Gly Arg Val Ala Leu

145 150 155 160

Val Lys Val Leu Asp Lys Trp Pro Leu Arg Ser Gly Gly Leu Gln Arg

165 170 175

Glu Gln Val Ile Ser Val Gly Ser Cys Val Pro Leu Glu Arg Asn Gln

180 185 190

Arg Tyr Ile Phe Phe Leu Glu Pro Thr Glu Gln Pro Leu Val Phe Lys

195 200 205

Thr Ala Phe Ala Pro Leu Asp Thr Asn Gly Lys Asn Leu Lys Lys Glu

210 215 220

Val Gly Lys Ile Leu Cys Thr Asp Cys Ala Thr Arg Pro Lys Leu Lys

225 230 235 240

Lys Met Lys Ser Gln Thr Gly Gln Val Gly Glu Lys Gln Ser Leu Lys

245 250 255

Cys Glu Ala Ala Ala Gly Asn Pro Gln Pro Ser Tyr Arg Trp Phe Lys

260 265 270

Asp Gly Lys Glu Leu Asn Arg Ser Arg Asp Ile Arg Ile Lys Tyr Gly

275 280 285

Asn Gly Arg Lys Asn Ser Arg Leu Gln Phe Asn Lys Val Lys Val Glu

290 295 300

Asp Ala Gly Glu Tyr Val Cys Glu Ala Glu Asn Ile Leu Gly Lys Asp

305 310 315 320

Thr Val Arg Gly Arg Leu Tyr Val Asn Ser Val Ser Thr Thr Leu Ser

325 330 335

Ser Trp Ser Gly His Ala Arg Lys Cys Asn Glu Thr Ala Lys Ser Tyr

340 345 350

Cys Val Asn Gly Gly Val Cys Tyr Tyr Ile Glu Gly Ile Asn Gln Leu

355 360 365

Ser Cys Lys Cys Pro Asn Gly Phe Phe Gly Gln Arg Cys Leu Glu Lys

370 375 380

Leu Pro Leu Arg Leu Tyr Met Pro Asp Pro Lys Gln Lys Ala Glu Glu

385 390 395 400

Leu Tyr Gln Lys Arg Val Leu Thr Ile Thr Gly Ile Cys Val Ala Leu

405 410 415

Leu Val Val Gly Ile Val Cys Val Val Ala Tyr Cys Lys Thr Lys Lys

420 425 430

Gln Arg Lys Gln Met His Asn His Leu Arg Gln Asn Met Cys Pro Ala

435 440 445

His Gln Asn Arg Ser Leu Ala Asn Gly Pro Ser His Pro Arg Leu Asp

450 455 460

Pro Glu Glu Ile Gln Met Ala Asp Tyr Ile Ser Lys Asn Val Pro Ala

465 470 475 480

Thr Asp His Val Ile Arg Arg Glu Thr Glu Thr Thr Phe Ser Gly Ser

485 490 495

His Ser Cys Ser Pro Ser His His Cys Ser Thr Ala Thr Pro Thr Ser

500 505 510

Ser His Arg His Glu Ser His Thr Trp Ser Leu Glu Arg Ser Glu Ser

515 520 525

Leu Thr Ser Asp Ser Gln Ser Gly Ile Met Leu Ser Ser Val Gly Thr

530 535 540

Ser Lys Cys Asn Ser Pro Ala Cys Val Glu Ala Arg Ala Arg Arg Ala

545 550 555 560

Ala Ala Tyr Asn Leu Glu Glu Arg Arg Arg Ala Thr Ala Pro Pro Tyr

565 570 575

His Asp Ser Val Asp Ser Leu Arg Asp Ser Pro His Ser Glu Arg Tyr

580 585 590

Val Ser Ala Leu Thr Thr Pro Ala Arg Leu Ser Pro Val Asp Phe His

595 600 605

Tyr Ser Leu Ala Thr Gln Val Pro Thr Phe Glu Ile Thr Ser Pro Asn

610 615 620

Ser Ala His Ala Val Ser Leu Pro Pro Ala Ala Pro Ile Ser Tyr Arg

625 630 635 640

Leu Ala Glu Gln Gln Pro Leu Leu Arg His Pro Ala Pro Pro Gly Pro

645 650 655

Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Ala Asp Met Gln Arg

660 665 670

Ser Tyr Asp Ser Tyr Tyr Tyr Pro Ala Ala Gly Pro Gly Pro Arg Arg

675 680 685

Gly Thr Cys Ala Leu Gly Gly Ser Leu Gly Ser Leu Pro Ala Ser Pro

690 695 700

Phe Arg Ile Pro Glu Asp Asp Glu Tyr Glu Thr Thr Gln Glu Cys Ala

705 710 715 720

Pro Pro Pro Pro Pro Arg Pro Arg Ala Arg Gly Ala Ser Arg Arg Thr

725 730 735

Ser Ala Gly Pro Arg Arg Trp Arg Arg Ser Arg Leu Asn Gly Leu Ala

740 745 750

Ala Gln Arg Ala Arg Ala Ala Arg Asp Ser Leu Ser Leu Ser Ser Gly

755 760 765

Ser Gly Gly Gly Ser Ala Ser Ala Ser Asp Asp Asp Ala Asp Asp Ala

770 775 780

Asp Gly Ala Leu Ala Ala Glu Ser Thr Pro Phe Leu Gly Leu Arg Gly

785 790 795 800

Ala His Asp Ala Leu Arg Ser Asp Ser Pro Pro Leu Cys Pro Ala Ala

805 810 815

Asp Ser Arg Thr Tyr Tyr Ser Leu Asp Ser His Ser Thr Arg Ala Ser

820 825 830

Ser Arg His Ser Arg Gly Pro Pro Pro Arg Ala Lys Gln Asp Ser Ala

835 840 845

Pro Leu

850

<210> 235

<211> 42

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 235

His Ala Arg Lys Cys Asn Glu Thr Ala Lys Ser Tyr Cys Val Asn Gly

1 5 10 15

Gly Val Cys Tyr Tyr Ile Glu Gly Ile Asn Gln Leu Ser Cys Lys Cys

20 25 30

Pro Asn Gly Phe Phe Gly Gln Arg Cys Leu

35 40

<210> 236

<211> 695

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 236

Met Ser Glu Gly Ala Ala Ala Ala Ser Pro Pro Gly Ala Ala Ser Ala

1 5 10 15

Ala Ala Ala Ser Ala Glu Glu Gly Thr Ala Ala Ala Ala Ala Ala Ala

20 25 30

Ala Ala Gly Gly Gly Pro Asp Gly Gly Gly Glu Gly Ala Ala Glu Pro

35 40 45

Pro Arg Glu Leu Arg Cys Ser Asp Cys Ile Val Trp Asn Arg Gln Gln

50 55 60

Thr Trp Leu Cys Val Val Pro Leu Phe Ile Gly Phe Ile Gly Leu Gly

65 70 75 80

Leu Ser Leu Met Leu Leu Lys Trp Ile Val Val Gly Ser Val Lys Glu

85 90 95

Tyr Val Pro Thr Asp Leu Val Asp Ser Lys Gly Met Gly Gln Asp Pro

100 105 110

Phe Phe Leu Ser Lys Pro Ser Ser Phe Pro Lys Ala Met Glu Thr Thr

115 120 125

Thr Thr Thr Thr Ser Thr Thr Ser Pro Ala Thr Pro Ser Ala Gly Gly

130 135 140

Ala Ala Ser Ser Arg Thr Pro Asn Arg Ile Ser Thr Arg Leu Thr Thr

145 150 155 160

Ile Thr Arg Ala Pro Thr Arg Phe Pro Gly His Arg Val Pro Ile Arg

165 170 175

Ala Ser Pro Arg Ser Thr Thr Ala Arg Asn Thr Ala Ala Pro Ala Thr

180 185 190

Val Pro Ser Thr Thr Ala Pro Phe Phe Ser Ser Ser Thr Leu Gly Ser

195 200 205

Arg Pro Pro Val Pro Gly Thr Pro Ser Thr Gln Ala Met Pro Ser Trp

210 215 220

Pro Thr Ala Ala Tyr Ala Thr Ser Ser Tyr Leu His Asp Ser Thr Pro

225 230 235 240

Ser Trp Thr Leu Ser Pro Phe Gln Asp Ala Ala Ser Ser Ser Ser Ser

245 250 255

Ser Ser Ser Ser Ala Thr Thr Thr Thr Pro Glu Thr Ser Thr Ser Pro

260 265 270

Lys Phe His Thr Thr Thr Tyr Ser Thr Glu Arg Ser Glu His Phe Lys

275 280 285

Pro Cys Arg Asp Lys Asp Leu Ala Tyr Cys Leu Asn Asp Gly Glu Cys

290 295 300

Phe Val Ile Glu Thr Leu Thr Gly Ser His Lys His Cys Arg Cys Lys

305 310 315 320

Glu Gly Tyr Gln Gly Val Arg Cys Asp Gln Phe Leu Pro Lys Thr Asp

325 330 335

Ser Ile Leu Ser Asp Pro Asn His Leu Gly Ile Glu Phe Met Glu Ser

340 345 350

Glu Glu Val Tyr Gln Arg Gln Val Leu Ser Ile Ser Cys Ile Ile Phe

355 360 365

Gly Ile Val Ile Val Gly Met Phe Cys Ala Ala Phe Tyr Phe Lys Ser

370 375 380

Lys Lys Gln Ala Lys Gln Ile Gln Glu Gln Leu Lys Val Pro Gln Asn

385 390 395 400

Gly Lys Ser Tyr Ser Leu Lys Ala Ser Ser Thr Met Ala Lys Ser Glu

405 410 415

Asn Leu Val Lys Ser His Val Gln Leu Gln Asn Tyr Ser Lys Val Glu

420 425 430

Arg His Pro Val Thr Ala Leu Glu Lys Met Met Glu Ser Ser Phe Val

435 440 445

Gly Pro Gln Ser Phe Pro Glu Val Pro Ser Pro Asp Arg Gly Ser Gln

450 455 460

Ser Val Lys His His Arg Ser Leu Ser Ser Cys Cys Ser Pro Gly Gln

465 470 475 480

Arg Ser Gly Met Leu His Arg Asn Ala Phe Arg Arg Thr Pro Pro Ser

485 490 495

Pro Arg Ser Arg Leu Gly Gly Ile Val Gly Pro Ala Tyr Gln Gln Leu

500 505 510

Glu Glu Ser Arg Ile Pro Asp Gln Asp Thr Ile Pro Cys Gln Gly Tyr

515 520 525

Ser Ser Ser Gly Leu Lys Thr Gln Arg Asn Thr Ser Ile Asn Met Gln

530 535 540

Leu Pro Ser Arg Glu Thr Asn Pro Tyr Phe Asn Ser Leu Glu Gln Lys

545 550 555 560

Asp Leu Val Gly Tyr Ser Ser Thr Arg Ala Ser Ser Val Pro Ile Ile

565 570 575

Pro Ser Val Gly Leu Glu Glu Thr Cys Leu Gln Met Pro Gly Ile Ser

580 585 590

Glu Val Lys Ser Ile Lys Trp Cys Lys Asn Ser Tyr Ser Ala Asp Val

595 600 605

Val Asn Val Ser Ile Pro Val Ser Asp Cys Leu Ile Ala Glu Gln Gln

610 615 620

Glu Val Lys Ile Leu Leu Glu Thr Val Gln Glu Gln Ile Arg Ile Leu

625 630 635 640

Thr Asp Ala Arg Arg Ser Glu Asp Tyr Glu Leu Ala Ser Val Glu Thr

645 650 655

Glu Asp Ser Ala Ser Glu Asn Thr Ala Phe Leu Pro Leu Ser Pro Thr

660 665 670

Ala Lys Ser Glu Arg Glu Ala Gln Phe Val Leu Arg Asn Glu Ile Gln

675 680 685

Arg Asp Ser Ala Leu Thr Lys

690 695

<210> 237

<211> 44

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 237

His Phe Lys Pro Cys Arg Asp Lys Asp Leu Ala Tyr Cys Leu Asn Asp

1 5 10 15

Gly Glu Cys Phe Val Ile Glu Thr Leu Thr Gly Ser His Lys His Cys

20 25 30

Arg Cys Lys Glu Gly Tyr Gln Gly Val Arg Cys Asp

35 40

<210> 238

<211> 115

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 238

Met Pro Thr Asp His Glu Glu Pro Cys Gly Pro Ser His Lys Ser Phe

1 5 10 15

Cys Leu Asn Gly Gly Leu Cys Tyr Val Ile Pro Thr Ile Pro Ser Pro

20 25 30

Phe Cys Arg Cys Val Glu Asn Tyr Thr Gly Ala Arg Cys Glu Glu Val

35 40 45

Phe Leu Pro Gly Ser Ser Ile Gln Thr Lys Ser Asn Leu Phe Glu Ala

50 55 60

Phe Val Ala Leu Ala Val Leu Val Thr Leu Ile Ile Gly Ala Phe Tyr

65 70 75 80

Phe Leu Cys Arg Lys Gly His Phe Gln Arg Ala Ser Ser Val Gln Tyr

85 90 95

Asp Ile Asn Leu Val Glu Thr Ser Ser Thr Ser Ala His His Ser His

100 105 110

Glu Gln His

115

<210> 239

<211> 42

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 239

His Glu Glu Pro Cys Gly Pro Ser His Lys Ser Phe Cys Leu Asn Gly

1 5 10 15

Gly Leu Cys Tyr Val Ile Pro Thr Ile Pro Ser Pro Phe Cys Arg Cys

20 25 30

Val Glu Asn Tyr Thr Gly Ala Arg Cys Glu

35 40

<210> 240

<211> 43

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 240

Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa

1 5 10 15

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa

20 25 30

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Arg Cys

35 40

Claims

1. Use of an antigen binding molecule that binds HER3 in the manufacture of a medicament for treating or preventing cancer in a subject, wherein the cancer comprises a cell containing an NRG gene fusion mutation that results in increased expression of a HER3 ligand, and wherein the antigen binding molecule comprises:

(i) a heavy chain variable region (VH) comprising the following CDRs:

as shown in SEQ ID NO: 41 of the amino acid sequence HC-CDR1

As shown in SEQ ID NO: 45 of the amino acid sequence shown in SEQ ID NO. 45-CDR 2

As shown in SEQ ID NO: 48, HC-CDR3 of the amino acid sequence set forth in SEQ ID NO; and

(ii) a light chain variable region (VL) comprising the following CDRs:

as shown in SEQ ID NO: 88, and LC-CDR1 of the amino acid sequence shown in

As shown in SEQ ID NO: 92, and LC-CDR2 of the amino acid sequence shown in seq id No. 92

As shown in SEQ ID NO: 95 of the amino acid sequence shown in seq id No. LC-CDR 3.

2. The use of claim 1, wherein the ligand of HER3 comprises an EGF-like domain of NRG.

3. The use of claim 1 or claim 2, wherein the NRG gene fusion is selected from the group consisting of CLU-NRG, CD-NRG, DOC-NRG, SLC 3A-NRG, RBPMS-NRG, WRN-NRG, SDC-NRG, RAB2 IL-NRG, VAMP-NRG, KIF 13-NRG, THAP-NRG, SMAD-NRG, MDK-NRG, TNC-NRG, DIP 2-NRG, MRPL-NRG, PARP-NRG, ROCK-NRG, DPYSL-NRG, ATP 1B-NRG, CDH-NRG, APP-NRG, AKAP-NRG, THBS-NRG, FOXA-NRG, PDE 7-NRG, RAB3 IL-NRG, CDK-NRG, BMPRIB-NRG, TNFRSF 10-NRG, nrph-NRG, and mca-SLC 12.

4. The use of claim 3, wherein the NRG gene fusion is selected from CLU-NRG1, CD74-NRG1, SLC3A2-NRG1 or VAMP2-NRG 1.

5. The use of claim 1 or claim 2, wherein the cancer comprises cells that express HER 3.

6. The use of claim 1 or claim 2, wherein the cancer is derived from lung, breast, head, neck, kidney, ovary, pancreas, prostate, uterus, gall bladder, colon, rectum, bladder, soft tissue, or nasopharynx.

7. The use according to claim 1 or claim 2, wherein the cancer is selected from lung cancer, breast cancer, head and neck cancer, kidney cancer, ovarian cancer, pancreatic cancer, prostate cancer, endometrial cancer, gallbladder cancer, cholangiocarcinoma, colorectal cancer, bladder carcinosarcoma, and neuroendocrine tumors.

8. The use of claim 7, wherein the cancer is selected from the group consisting of non-small cell lung cancer, lung adenocarcinoma, aggressive lung mucus adenocarcinoma, lung squamous cell carcinoma, malignant epithelial carcinoma of the breast, invasive carcinoma of the breast, squamous cell carcinoma of the head and neck, clear cell carcinoma of the kidney, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, malignant epithelial carcinoma of the prostate, sarcoma of the uterus, urothelial bladder cancer, soft tissue sarcoma, and neuroendocrine tumor of the nasopharynx.

9. The use of claim 1 or claim 2, wherein the antigen binding molecule comprises:

a VH region comprising the following framework regions:

as shown in SEQ ID NO: HC-FR1 of amino acid sequence shown in 53

As shown in SEQ ID NO: HC-FR2 of amino acid sequence shown in 59

As shown in SEQ ID NO: 66 of the amino acid sequence shown in formula II HC-FR3

As shown in SEQ ID NO: 71 of the amino acid sequence shown in HC-FR 4.

10. The use of claim 1 or claim 2, wherein the antigen binding molecule comprises:

a VL region comprising the following framework regions:

as shown in SEQ ID NO: 104 of the amino acid sequence of LC-FR1

As shown in SEQ ID NO: 110 of the amino acid sequence shown in LC-FR2

As shown in SEQ ID NO: 120 of the amino acid sequence LC-FR3

As shown in SEQ ID NO: 125, or a pharmaceutically acceptable salt thereof.

11. The use according to claim 1 or claim 2, the antigen binding molecule comprising the amino acid sequence as set forth in SEQ ID NO: 36, or a VH region of the amino acid sequence shown in figure 36.

12. The use according to claim 1 or claim 2, the antigen binding molecule comprising the amino acid sequence as set forth in SEQ ID NO: 83 of the amino acid sequence set forth in seq id no.

13. The use of claim 1 or claim 2, wherein the antigen binding molecule comprises the amino acid sequence as set forth in SEQ ID NO: 171.

14. The use of claim 1 or claim 2, wherein the antigen binding molecule comprises the amino acid sequence as set forth in SEQ ID NO: 177, or a pharmaceutically acceptable salt thereof.